Microarrays and their manufacture by slicing

ABSTRACT

Microarrays are prepared by using a separate fiber for each compound being used in the microarray. The fibers are bundled and sectioned to form a thin microarray that may be glued to a backing.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The instant application is a continuation-in-part of patentapplication Ser. No. 09/628,339 filed Jul. 28, 2000 which is acontinuation-in-part of patent application Ser. No. 09/482,460 filedJan. 13, 2000 which is a continuation-in-part of application Ser. No.60/146,653 filed Jul. 30, 1999, the contents of which are incorporatedherein in entirety.

FIELD OF THE INVENTION

[0002] The instant invention relates to microarrays containingbioreactive molecules, uses thereby and methods for manufacture thereof.The arrays are constructed by sectioning bundles of tubules or rods,each containing unique reactants to produce large numbers of identicalarrays.

BACKGROUND OF THE INVENTION

[0003] A microarray is essentially a two-dimensional support or sheetwherein different portions or cells (sectors) of the support or sheetcarry different biomolecules or elements, such as, nucleotides,polynucleotides, peptides, polypeptides, saccharides or polysaccharides,bound thereto. Microarrays are similar in principle to other solid phasearrays except that assays involving such microarrays are performed on asmaller scale, allowing many assays to be performed in parallel.Microarrays have been used for a number of analytical purposes,typically in the biological sciences.

[0004] Biochemical molecules on microarrays have been synthesizeddirectly at or on a particular cell (sector) on the microarray, orpreformed molecules have been attached to particular cells (sectors) ofthe microarray by chemical coupling, adsorption or other means. Thenumber of different cells (sectors) and therefore the number ofdifferent biochemical molecules being tested simultaneously on one ormore microarrays can range into the thousands. Commercial microarrayplate readers typically measure fluorescence in each cell (sector) andcan provide data on thousands of reactions simultaneously thereby savingtime and labor. A representative example of a patent in the field isU.S. Pat. No. 5,545,531.

[0005] Currently, two dimensional arrays of macromolecules are madeeither by depositing small aliquots on flat surfaces under conditionswhich allow the macromolecules to bind or be bound to the surface, orthe macromolecules may by synthesized on the surface usinglight-activated or other synthetic reactions. Previous methods alsoinclude using printing techniques to produce such arrays. Some methodsfor producing arrays have been described in “Gene-ExpressionMicro-Arrays: A New Tool for Genomics”, Shalon, D., in FunctionalGenomics; Drug Discovery from Gene to Screen, IBC Library Series,Gilbert, S. R. & Savage, L. M. , eds., International BusinessCommunications, Inc., Southboro, Mass., 1997, pp 2.3.1.-2.3.8; “DNAProbe Arrays: Accessing Genetic Diversity”, Lipshutz, R. J. , inGilbert, S. R. & Savage, L. M. , supra, pp 2.4.1.-2.4.16; “Applicationsof High-Throughput Cloning of Secreted Proteins and High-DensityOligonucleotide Arrays to Functional Genomics”, Langer-Safer, P. R. , inGilbert, S. R. & Savage, L. M. , supra; Jordan, B. R. , “Large-scaleexpression measurement by hybridization methods: from high-densities to“DNA chips”, J. Biochem. (Tokyo) 124: 251-8, 1998; Hacia, J. G. , Brody,L. C. & Collins, F. S. , “Applications of DNA chips for genomicanalysis”, Mol. Psychiatry 3: 483-92, 1998; and Southern, E.M., “DNAchips: Analyzing sequence by hybridization to oligonucleotides on alarge scale”, Trends in Genetics 12: 110-5, 1996.

[0006] Regardless of the technique, each microarray is individually andseparately made, typically is used only once and cannot be individuallyprecalibrated and evaluated in advance. Hence, one depends on thereproducibility of the production system to produce error-free arrays.Those factors have contributed to the high cost of currently producedbiochips or microarrays, and have discouraged application of thetechnology to routine clinical use.

[0007] For scanning arrays, charged coupled device (CCD) cameras can beused. The cost of those devices has declined steadily, with suitablecameras and software now widely available. Such devices generally detectlight sources or light absorbance. In one proposed variation, an arrayis located at the ends of a bundle of optical fibers with the nucleicacid or antibody/antigen attached to the other end of the optical fiber.Detection of fluorescence then may be performed through the opticalfiber, for example, see U.S. Pat. No. 5,837,196.

[0008] Fiber optical arrays can be produced in which glass or plasticfibers are aligned in parallel in such a manner that all remainparallel, and an optical image may be transmitted through the array.Parallel arrays also may be made of hollow glass fibers, and the arraysectioned normal to the axis of the fibers to produce channel platesused to amplify optical images. Such devices are used for night visionand other optical signal amplification equipment. Channel plates havebeen adapted to the detection of binding reactions (U.S. Pat. No.5,843,767, not prior art) with the individual holes being filled aftersectioning of the channel plate bundle, and discrete and separateproteins or nucleic acids being immobilized in separate groups of holes.

[0009] Hollow porous fibers have been used for dialysis of biologicalsamples, for example, in kidney dialyzers and for water purification.Methods for aligning the fibers in parallel arrays, for impregnating thevolume between the fibers with plastic, and for cutting the ends of sucharrays have been described (see, for example, U.S. Pat. No. 4,289,623).

[0010] Immobilized enzymes have been prepared in fiber form from anemulsion as disclosed, for example, in Italy Pat. No. 836,462.Antibodies and antigens have been incorporated into solid phase fibersas disclosed in U.S. Pat. No. 4,031,201. A large number of otherdifferent immobilization techniques are known in the fields of solidphase immunoassays, nucleic acid hybridization assays and immobilizedenzymes, see, for example, Hermanson, G. T. , Bioconiugate Techniques.Academic Press, New York. 1995, 785 pp; Hermanson, G. T. , Mallia, A. K.& Smith, P. K. Immobilized Affinity Ligand Techniques. Academic Press,New York, 1992, 454 pp; and Avidin-Biotin Chemistry: A Handbook. D.Savage, G. Mattson, S. Desai, G. Nielander, S. Morgansen & E. Conklin,Pierce Chemical Company, Rockford Ill. , 1992, 467 pp.

[0011] Currently available biochips include only one class ofimmobilized reactant, and perform only one class of reactions. For manytypes of clinical and other analyses, there is a need for chips that canincorporate reactants immobilized in different ways in one chip.

SUMMARY OF THE INVENTION

[0012] The instant invention relates to a method for producing rods ortubules, each containing a different entrapped or attached biologicalagent of interest; for arranging and keeping the rods or tubules inparallel bundles; optionally, for impregnating or embedding the bundleswith a sectionable adhesive material; optionally, for checking that allelements of the bundle maintain a constant arrangement or patternthroughout the length of the bundle after impregnation; for sectioningthe bundle to produce large numbers of identical arrays or chips; andfor performing a variety of different quantitative biochemical analyseson individual arrays or chips based on, for example, enzymaticactivities, immunochemical activities, nucleic acid hybridization andsmall molecule binding under conditions yielding, for example,fluorescence, optical absorbance or chemiluminescence signals, foracquiring images of the signals which can be processed electronicallyand compared to produce clinically and experimentally useful data.

[0013] In a further, the invention relates to long filaments or tubesthat contain, are coated with, or have an agent of interest embeddedtherein, and methods for manufacture thereof.

[0014] In another aspect, the invention relates to a device comprising asubstrate or solid phase having at least two (2) opposing major surfacesand multiple discrete channels extending to at least the two opposingsurfaces. Further, the channels may comprise a first and second bindingreagent immobilized on the walls of a first and second group ofchannels. In a related aspect, the binding reagents may or may not beidentical.

[0015] In a further aspect, the invention relates to a device comprisinga rigid support that is integral to a substrate or solid phase or isbonded to a substrate or solid phase. In a related aspect, the substrateor solid phase has a flat surface and a plurality of structuresprojecting away from the plane of the solid phase. Moreover, suchstructures may be permeable or impermeable to fluids.

[0016] The invention also relates to methods for arranging the fibers toform bundles in which the position of each fiber relative to all othersis retained throughout the bundle length.

[0017] The invention further relates to means and methods for attachingor gluing all of the fibers together over the entire length thereof.

[0018] In a related aspect, the invention relates to the preparation ofmicroarrays wherein the elongated filaments or tubes are bundledtogether and cut transversely many times at short intervals to yieldcross sectional slices thereof to form microarrays and a microarray soprepared.

[0019] A further aspect of the invention is the inclusion of markerswhich are either integral with the tubes or fibers or are contained inthe media contained in hollow fibers which allow the fibers to bedistinguished along the entire length thereof.

[0020] An additional aspect of the invention includes means forilluminating fibers individually at one end of a bundle, and identifyingthe other end by photoelectric means to confirm the integrity of thefiber arrangement.

[0021] In another aspect, the instant invention relates to forming afiber containing an agent of interest, or means for immobilizing one ora class of agents of interest thereto.

[0022] In an additional aspect, the invention relates to means forembedding or attaching whole or fragments of biological cells, tissuesor infectious agents to fibers or tubules in such a manner that thebiologicals are exposed on the cut end of each fiber of tubule.

[0023] In another aspect of the invention, the array consists of tubulescontaining gel or other polymerizing materials that adhere to the tubingwalls.

[0024] In a further aspect of the invention, agents of interest areattached to the polymerizing or suspending medium in the lumen of smalltubes.

[0025] In yet another aspect of the invention, the agents of interestare attached to particles that are suspended in a polymerizing medium,which suspension is used to fill tubules used to make array bundles andarrays.

[0026] The invention further relates to a method for the large scaleproduction of identical flat two-dimensional arrays of immobilizednucleic acid-based agents for use in nucleic acid sequencing, in theanalysis of complex mixtures of ribonucleic acids (RNA's) anddeoxyribonucleic acids (DNA's), and in the detection and quantificationof other analytes including proteins, polysaccharides, organic polymersand low molecular mass analytes, by sectioning long bundles of fibers ortubes containing same.

[0027] In a related aspect, the invention relates to exploitingmicroarrays for mass screening of large numbers of samples from one to alarge number of agents of interest.

[0028] In another aspect of the instant invention, one may performquality control assays on each fiber after manufacture, so that onlyfully functional fibers are included in a fiber bundle.

[0029] In a further related aspect, the invention relates to thedevelopment of sets of tests on different chips or microarrays done inoptionally branching sequence, which reduces the cost, delay andinconvenience of diagnosing human diseases, while providing complex dataordinarily obtained by time-consuming sequential batteries ofconventional tests.

[0030] In still another aspect, the invention relates to the fabricationof identical arrays that are sufficiently inexpensive to allow severalidentical arrays to be mounted on the same slide or test strip, andcross-compared for quality control purposes.

[0031] In a still further aspect, the invention relates to theincorporation of a non-fluorescent dye or other light absorbing materialin the substance of the array to control the depth to which light usedto excite fluorescence penetrates the array, thereby controlling thedepth to which fluorescence analytes are detected, and insuring thatfluorescent analytes which diffuse too deeply into the content of thecells, and therefore do not diffuse out, are not detected.

[0032] In another aspect, the invention relates to methods fordetermining that tubules are completely full of support media, and lackvoids or air bubbles.

[0033] In a further aspect, the invention relates to methods andapparatus for completely filling small tubes with a supporting mediumusing hydrostatic force or centrifugal force.

[0034] In an additional aspect, the invention relates to thereproducible manufacture of biochips or microarrays for bioanalysis.

[0035] In a further aspect, the invention relates to the design andproduction of arrays, which are specifically designed to detect anddiagnose a specific disease.

[0036] In yet another aspect, the instant invention relates to multiwellplates and methods for manufacture thereof.

[0037] In yet a further aspect, the invention relates to increasing thedynamic range of multiple-parallel assays by providing means for makingserial measurements of fluorescence or absorbance over time, and fordetermining the rate of change of fluorescence or absorbance in eachelement of the array over time.

[0038] It is an additional aspect of the invention to produce biochipsthat are inexpensive and sufficiently standardized to allow more thanone to be used for each analysis, and for controls and standards to berun routinely and simultaneously in parallel. For added qualityassurance, sections from different portions of the bundle or differentends may be used. One way of sectioning from different portions of thebundle is to cut or bend the bundle in the middle and align the twohalves to form a single larger bundle thereby producing a section whereeach fiber is represented twice.

[0039] In a further aspect, the invention relates to the production ofchips in which the array elements or cells (sectors) may differ from oneanother in the composition of the tubes, supporting medium,immobilization surface, or the class of agent of interest may bedifferent in different cells (sectors).

[0040] In an additional aspect, the invention relates to the productionof chips in which different types of reactions may be carried out at thesurface of each cell (sector) of the array, with the reactions includingimmunological, enzymatic or hybridization reactions.

[0041] A further aspect of this invention relates to the production ofsubarrays of fibers or tubules adhering together to form one-dimensionalribbon-like arrays which may be separately stored. The “ribbons” may besubject to quality control analysis before being assembled intotwo-dimensional arrays. Different one-dimensional arrays may be used toassemble different arrays, thus providing the option of producingcustom-made arrays to meet specific research and clinical requirements.

[0042] The invention further relates to the development of multipleparallel chip-based methods involving continuously increasingtemperature such that temperature sensitive reactions may be carried outat physiological temperatures, followed by an increase in temperature toallow hybridization reactions to occur.

[0043] In a still further aspect, the invention relates to preparinglibraries of compounds with each fiber containing one of the compounds.The array may be used to screen simultaneously all of the compounds fora particular chemical or biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a schematic of intermediate products in the process forproducing microarrays.

[0045]FIG. 2 is a schematic of an individual tubule containing beadswith immobilized ligands embedded in a gel.

[0046]FIG. 3 is a schematic of an individual tubule containing a gelwith ligands attached to the gel.

[0047]FIG. 4 is a schematic of an array with ligands attached to theinner walls of cells, and with means for closing off one surface of thearray to form a set of microwells.

[0048]FIG. 5 is a schematic of a means for insuring that all fibers aremaintained in their correct pattern before the bundle is sliced.

[0049]FIG. 6 is a schematic of means for identifying arrays.

[0050]FIG. 7 is a schematic for scanning an array.

[0051]FIG. 8 displays an alternative way of forming a fiber bundle.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The terms “binding component”, “molecule of interest”, “agent ofinterest”, “ligand” or “receptor” may be any of a large number ofdifferent molecules, biological cells or aggregates, and the terms areused interchangeably. Each binding component is immobilized at a cell,sector, site or element of the array and binds to an analyte beingdetected. Therefore, the location of an element or cell containing aparticular binding component determines what analyte will be bound.Proteins, polypeptides, peptides, nucleic acids (nucleotides,oligonucleotides and polynucleotides), antibodies, ligands, saccharides,polysaccharides, microorganisms such as bacteria, fungi and viruses,receptors, antibiotics, test compounds (particularly those produced bycombinatorial chemistry), plant and animal cells, organdies or fractionsof each and other biological entities may each be a binding component ifimmobilized on the chip. Each, in turn, also may be considered asanalytes if same bind to a binding component on a chip.

[0053] When a molecule of interest has a high molecular weight, it isreferred to as a “macromolecule”. In terms of some biopolymers, the highmolecular weight refers to greater than 100 amino acids, nucleotides orsugar molecules long.

[0054] The term “bind” includes any physical attachment or closeassociation, which may be permanent or temporary. Generally, aninteraction of hydrogen bonding, hydrophobic forces, van der Waalsforces, covalent and ionic bonding etc., facilitates physical attachmentbetween the molecule of interest and the analyte being measuring. The“binding” interaction may be brief as in the situation where bindingcauses a chemical reaction to occur. That is typical when the bindingcomponent is an enzyme and the analyte is a substrate for the enzyme.Reactions resulting from contact between the binding agent and theanalyte are also within the definition of binding for the purposes ofthe present invention.

[0055] The term “cells”, “sectors”, “sites” or “elements” in the instantapplication refers to a unit component of an array identified by aunique address, which generally differs from other cells, sectors, sitesor elements by content as well as location. Biological cells generallyare referred to by type, e.g. microorganisms, animal and plant cells.

[0056] The term “fibers” includes both filaments and hollow capillarystructures. Filaments or rods may be solid strands of monolithic, porousor composite forms, or aggregate forms. Pluralities, typically a largenumber, of fibers are bound adjacent to each other in ribbons or bundlesto form a “fiber bundle.” A fiber bundle may constitute a portion of theactual bundle being used such as ribbon. The cross-section of the fibersmay be of any shape, such as round, triangular, square, rectangular orpolygonal.

[0057] The term “surface” refers to the exterior boundaries of anobject.

[0058] The term “particle” includes a large number of insolublematerials of any configuration, including spherical, thread-like,brush-like and many irregular shapes. Particles are frequently porouswith regular or random channels inside. Examples include silica,cellulose, Sepharose beads, polystyrene (solid, porous and derivitized)beads, controlled-pore glass, gel beads, sols, biological cells,subcellular particles, microorganisms (protozoans, bacteria, yeast,viruses, etc.) micelles, liposomes, cyclodextrins, two phase systems(e.g. agarose beads in wax) etc. and other structures which entrap orencapsulate a material. Particularly preferred are recombinant hosts andviruses that express the protein of interest. Even certain highmolecular weight materials, such as, polymers and complexes, may serveas immobilizing structures that would constitute a “particle”.

[0059] The term “sintering” refers to the adhesion of the surfaces ofthe fibers without actually melting the whole fiber. Sintering may bechemical or thermal and may even involve a self adhesive component thatmay be activatable.

[0060] The terms “arrays” and “microarrays” are used somewhatinterchangeably differing only in general size. The instant inventioninvolves the same methods for making and using either. Each arraytypically contains many cells (typically 100-1,000,000+) wherein eachcell is at a known location and contains a specific component ofinterest. Each array therefore contains numerous different components ofinterest.

[0061] In a related aspect, “device” is used to describe both arrays andmicroarrays, where the array or microarray may comprise other definedcomponents including surfaces and points of contact between reagents.

[0062] Further, “substrate” is also a term used to describe surfaces aswell as solid phases which may comprise the array, microarray or device.

[0063] The instant invention makes microarrays, “chips” or “biochips” bysectioning bundles of small plastic rods, fibers, tubes or tubulescontaining immobilized binding component, including biological moleculesand entities such as nucleic acid fragments, nucleotides, antigens,antibodies, proteins, peptides, carbohydrates, ligands, receptors, drugtargets, biological cells or subfractions thereof (e.g. ground-up cells,organelles, solvent extract etc.), infectious agents or subfractionsthereof, drugs, toxic agents or natural products. Embedding media maybe, in the instant invention, polymerized or solidified in small tubes,or may be cast into rods or sheets.

[0064] The tubes may be of material such as glass, metal, ceramic orplastic. The immobilized binding components, e.g. nucleic acids,proteins, cells etc., may be coated on the inside or outside of themicrotubes, contained in a gel in the microtubes, or attached to orembedded in small particles or beads which fill the tubes. The particlesor beads may be a component of a gelling material or can be separatecomponents such as latex beads made of a variety of synthetic plastics(polystyrene etc.). When the individual fibers are solid rods orfilaments, the agent of interest is incorporated on or in the plasticbefore the filament is cast, extruded or pulled through a die. Eachsection cut constitutes a microarray for use in various binding assays.

[0065] A key aspect of the invention, which provides an economicadvantage, is that the fibers or tubules are prepared using only methodsproviding a functionality stable to long term storage are used. Unlikeother methods involving protein containing liquids which must beprepared fresh each time, immobilized proteins in relatively dry formremain stable for great lengths of time, often without refrigeration.

[0066] The preparation of each component of a future microarrayseparately in/on a fiber permits one to assay for and evaluate thefunctionality or reactivity of each component before being incorporatedin an array. Both the spotting technique and the in situ synthesistechnique do not permit testing before completion. Furthermore, qualitycontrol checks can sample only a small portion of such microarrays,which is unlike the instant invention where each fiber may be tested.

[0067] Various aspects of the invention are illustrated in FIGS. 1-7.

[0068] General principles are illustrated in FIG. 1 where rod or tube 1incorporates an agent of interest. The rods or tubes may be bonded intoa flat parallel array 2, and multiple flat arrays then are bonded intothe multiple parallel bundle 3. Alternatively, the bundle 3 may beconstructed in one step from a series of rods 1. The end of bundle 3 iscut or sectioned to yield the final array 4 that contains one smallsection 5 of each rod or tube in the entire bundle. By making a longbundle 3, and cutting very small sections 4, a very large number ofidentical arrays or chips are formed. For example, if bundle 3 is ameter long, and the sections are 10 microns thick, 100,000 identicalchips may be produced.

[0069] In the case of hollow glass fibers, such as those in channelplates, the hollow fibers may be filled with gels or particles includingimmobilized reactants, and the entire bundle sawed into arrays.

[0070] The rods or tubules comprising the sectioned bundle fall into atleast eight classes, with subdivisions of each.

[0071] A first class is composed of solid rods or filaments with theimmobilized binding component being part of the composition of the rodor filament. The agent of interest in the instant invention may comprisea very broad range of chemicals, complexes, tissues, biological cells orfractions thereof. Nucleic acids, sugars, proteins, which may bemodified or coated with detergents to enhance solubility in organicsolvents, and a wide range of organic compounds can be incorporated intopolymerizing mixtures such as those used to produce plastics.Oligonucleotides and nucleic acids are soluble in methylene chloride,for example, and hence may be included in acrylics duringpolymerization.

[0072] A number of polymerizing embedding agents have been developed forhistological and histochemical studies, some of which are listed inTable 1, together with data on composition, curing temperature, solventused and viscosity. TABLE I CURE RESIN* TYPE TEMP. SOLVENT VISCOSITYDurcupan —   40° C. Water Medium Nanoplast Melamine   60° C. Water LowQuetol 651 Epoxy   60° C. Water Low London Resin Gold Acrylic, −25° C.Water, EtOH Low UV Curing Lowicryl K4M Acrylic, −35° C. Water, EtOH LowPolar UV Curing Lowicryl Monostep Acrylic, −35° C. Water, EtOH LowK4Mpolar UV Curing Lowicryl K11 Polar Acrylic −60° C. EtOH Low UV CuringJB-4 GMA RT Water Low JB-4 Plus Methacrylate RT Water Low ImmunoBed GMART Water Low PolyFreeze Polyol −15° C. Water Low

[0073] Other methods for impregnating a solid fiber include mobilizingthe agent of interest through the matrix of the solid fiber using anelectromotive force.

[0074] In one embodiment, the microarrays are produced by diffusion andentrapment after polymerization of the strands. An element of amicroarray is formed by preforming a polymeric strand, thenincorporating a biological target molecule into the strand by a methodincluding, but not limited to, diffusion from a solution containing thebiological target molecule. Such a method of incorporating labilebiological target molecules into polymeric avoids harsh conditions ofpolymerization, such as heat, presence of free radicals etc. that mightalter a biological target molecule. Further, such a method envisagesentrapping the biological target molecule within the polymeric strandwhile concomitantly preventing subsequent diffusion of the biologicaltarget molecule out of the strand. For example, a polymeric strand ofmaterial can be prepared from a material such as, but not limited to,ImmunoBed (Polysciences, Warrington, Pa.), polyacrylamide, agarose etc.Further, a mixture of monomeric substances can be mixed with anentrapping agent, such as but not limited to, protein-biotin complexes.The mixture can then be introduced into tubing (e.g., polyethylene) andallowed to polymerize. A strand thus formed can be extruded from thetubing mold and placed in a solution containing a biological targetmolecule of interest. In one embodiment, the biological target moleculeof interest will be conjugated to a biotin-binding protein such asstreptavidin. However, other binding pairs, which are known in the art,are also envisaged for this purpose (e.g., antibody-antigen, nucleicacid-nucleic acid, protein-protein, protein-nucleic acid,receptor-ligand, lectin-antibody, cell-cell, etc). The strand is allowedto remain in contact with the solution for a period of time, at atemperature that is consistent with maintenance of biological activity.In one embodiment, the temperature range is between about 4° C. to about70° C., more preferably about 4° C. to about 4° C. The temperature ischosen based on the solidifying properties of the matrix and thethermolabile properties of the agent of interest.

[0075] The period of time of contact between the strand and the solutionfor optimal incorporation of the biological target molecule into thestrand will depend on many factors, including, but not limited to,porosity of the strand, molecular size of the biological targetmolecule, concentration of the biological target molecule, temperatureetc. After the strand is loaded with biological target molecule, thestrand is washed with buffer and aligned with other strands to form anarray.

[0076] A second class of fibers is not homogeneous and the polymerizingor gelling material also may contain solid structural elements such asfilaments, branched elements etc., to further strengthen the gel andalso may provide attachment sites for the agent of interest. Thus, theadded components serve to strengthen the gel and may provide attachmentsites for inclusions including dendrimer branched polynucleic acids,branched or crosslinked polymeric materials, metal or glass fibers.Threads, yarn-like configurations and brush-like configurations ofstructural elements may be cast into the length of the fiber to providestrength and to allow the fiber to be handled or dried more easily. Thestructural elements may serve as the immobilizing component in the fiberfor a desired binding component.

[0077] Thus, it is technically feasible to produce long fibers ofacrylic or other plastics each containing a different agent of interestusing currently available extrusion technology in the instant invention.The cut end of the fibers may be treated briefly with dilute solvents toexpose active groups.

[0078] A third class of fibers includes extruded or cast plastic, whichincludes a second phase. The second phase may be in the form of, forexample, hydrocarbon, aqueous or fluorocarbon microdroplets, particlesof sugars or other water soluble materials, or inorganic particles suchas calcium carbonate particles, which can be dissolved in dilute acid toreveal active groups. Brief exposure of the cut surface of a chip to asolvent will dissolve some of the inclusions, increasing the surfacearea of the support plastic containing the agents of interest. Inanother embodiment, the material between the fibers can be removed,increasing the available surface on the exterior of the fiber forinteraction between target and agent of interest.

[0079] In a related aspect, each fiber may contain two or more materialspossessing different properties such as, but not limited to,polyethylene fiber and an acrylamide gel with the protein. The materialsmay be attached by a cyanoacrylate adhesive. Likewise, for the glueholding the fibers together, the glue may be made of a differentmaterial. In a related aspect, such mixed fibers may comprise the sameor different protein immobilized in a matrix comprising one or moreheterogeneous materials.

[0080] Solid plastics also can be prepared which incorporate polystyrenelatex or other plastic particles to which proteins or nucleic acids areattached. Conditions can be arranged such that the supporting plastic iseroded to a depth of a few microns to reveal active subparticlesurfaces, and do not dissolve the supporting plastic latex beads. Forexample, proteins derivatized with fluorinated groups attach strongly toTeflon® microparticles. Such derivatized Teflon® particles in, forexample, an acrylic plastic or other suitable embedding medium, can bepartially exposed at the plastic surface by a dilute acrylic solvent,composed, for example, of methylene chloride and ethyl alcohol.Alternatively, the particles may be embedded in a porous matrix.

[0081] The beads to which agents of interest are attached may be porousgel beads used in chromatography such as Sephadex, Biogels and others,or solid beads such as are used in chromatography. A variety of methodsfor derivitizing such support structures and for attaching polypeptides,proteins, nucleic acids, polynucleotides, saccharides, polysaccharidesand small molecules thereto have been developed and are known to thoseskilled in the arts. The construction of such tubules is illustrated inFIG. 2 where tubule 6 is comprised of tube 7 containing gel 8 whichsupports particles 9. An end view 10, and enlarged view 11 of the tubuleshows exposed particles 12 at the cut end. Area 13 is shown additionallyenlarged at 14 to illustrate the presence of immobilized reactants 15 onthe surface of the exposed particles 12.

[0082] Note that all rods described can be cast with a string or threadthrough the center thereof to increase strength, and to make the rodseasier to handle.

[0083] A fourth class of fibers is prepared by sintering glass orplastic beads to form a porous material with a high surface to massratio. Such material is conventionally made from glass,polytetrafluoroethylene (PTFE) (Teflon®), Teflon® AF, polyethylene,polypropylene, can be manufactured from polystyrene and from a varietyof other plastics. Heat, pressure or exposure to solvent vapors cansinter plastics. The sintered material can be derivatized in sheets orin cut rods. Polystyrene is convenient from the point of view ofcoupling agents of interest thereto. For polystyrene derivatization,methods that allow attachment of proteins by the amino groups, carboxylgroups, or sulfhydryl groups thereof, have been described. Teflon® canbe activated using solutions of metallic sodium in an organic solventproducing groups to which other plastics will adhere, and then may bederivatized. Polyethylene and polystyrene can be activated by coronaplasma discharge or by electron beam radiation. An advantageous approachis to make sintered composites of polystyrene and polyethylene. Nylonbeads also can be sintered and derivatized. Other sintered materials areknown or are under development, many of which will find applicationhere.

[0084] Molecules of interest may be attached to the solid materialseither before or after sintering. For attachment of the ligands, therods may be soaked in tubes containing the substance to be attached orthe rods may be coiled up inside a hollow bowl centrifuge rotor havingthe general configuration of a zonal rotor (see Anderson, N. G., Natl.Cancer Inst. Monograph No. 21), but which may be centrifugally drained.The solution of the substance to be attached then is centrifuged firstinto the sintered mass, and then out of it, followed by washing asnecessary. The sintered rods then may be dried, coated with a suitableadhesive, assembled into a bundle and sectioned.

[0085] Alternatively, the beads with agents and items of interestattached thereto may be extruded under pressure to form rods that thenmay be sintered together. The assembled tubes may be held together witha variety of cements or polymerizeable plastics. The outside of thetubes may be altered or treated so that cements or polymerizeableplastics will adhere thereto.

[0086] A fifth class of fibers is comprised of hollow impermeabletubules typically formed from plastics including, but not limited to,polyethylene, polypropylene, Teflon® or polyvinyl chloride, and isfilled completely with a gel or other polymerizing material to whichagents of interest are attached directly. The external surfaces of thetubes may be modified chemically or physically to accept adhesives usedto bind the bundled tubes together. The internal surface also may bemodified so that the gel or polymerizing mixture introduced into thetubes will adhere, preferably by covalent attachment. Acrylamidederivatives may be linked to the wall to make an acrylamide gel adhere,while gelatin, agar, or agarose derivatives may be attached similarly tolink with the respective gels. Methods for linking agents of interest,such as, proteins and nucleic acids, to linear acrylamide, gelatin andagarose are well known, and the derivatized molecules can beincorporated into the gels used for casting. Acrylamide can be made togel at room temperature either chemically or using photoactivation,while low temperature-gelling Sepharose is available. Gelatin setsslowly and at temperatures below ambient. The polymers used to fill thetubes are typically homogeneous, but may contain agents of interest,which become attached to the polymerizing medium. Examples includecovalent attachment of proteins to short acrylamide chains that becomeincorporated into acrylamide gels and proteins covalently linked togelatin. Thus, gels are available or can be produced which containlabile biomolecules without exposing them to denaturing temperatures.The structure of such tubes is illustrated in FIG. 3 where tube 16 isfilled with a cross-linked gel 17 to which are attached agents ofinterest 18. A side view 19 and end view 21 of a sectioned tubeillustrates the availability of immobilized agents 21.

[0087] Arrays prepared using hollow fibers may have the interior of thefibers coated with biomolecules either covalently or in suitable polymercoatings, or in gels before the array is assembled. Isocyanate polymers,such as oxyethylene-based diols or polyols wherein most if not all ofthe hydroxyl groups thereof carry polyisocyanate groups are suitable.Some such polymers can be comprised of polyurea/urethane polymers. Thepolymers are well hydrated and fall in the category of hydrogels.Suitable starting materials include triols, such as glycerol,trimethylpropane and triethanolamine, tetrols and polyethylene glycols.Suitable polyisocyanates include diisocyanates and such. Thepolyisocyanates can be aromatic, aliphatic or cycloaliphatic. (Braatz etal., U.S. Pat. No. 5,169,720 and Braatz, J. Biomaterials Applications9:71-96 (1994)). Alternatively, a bundled array may be positioned sothat individual hollow fibers may be filled with biopolymers insolutions that gel prior to sectioning.

[0088] A sixth class of fibers or tubes includes empty impermeable tubeswith molecules of interest attached to the inner surface, but otherwiseempty or made empty. As illustrated in FIG. 4, the sectioned chip 22 iscomprised of sectioned plastic tubes 23 embedded in supporting plastic24, with the agent of interest 25 attached to the inner walls of thetubes, leaving the center 26 open. The result 27, seen in side section,has sectioned plastic tube 23, immobilized agent 25, yielding open holes26, and all held together by supporting material 24. The chips may beconsidered as ultramicrotiter plates and may be used for flow throughanalysis based on, for example, immobilized affinity ligand techniques(Hermanson et al., Immobilized Affinity Ligand Techniques, AcademicPress, 1992, p 407), for polymerase chain reaction (PCR) amplificationof immobilized oligonucleotides, or for other detection reactions andthe like that can be accomplished at that scale, as described, forexample, in U.S. Pat. No. 5,843,767. When the tubes are made of Teflon®with the internal or external surfaces treated to become hydrophilic,the cut ends will remain hydrophobic. When a hydrophilic test solutionis spread across the surface of the chip, the solution tends to flowinto the holes in self-controlling volumetric amounts, and, if the totalamount of fluid is controlled properly, tends not to affect adjacentcells. The upper and lower surfaces then can be sealed with a suitableadhesive tape and the whole subjected to reactions, for example, forpolymerase chain reaction amplification of DNA. Alternatively thesandwiched structure 32 including chip 33 of FIG. 4 may employ twopieces of material such as glass or quartz 34 to seal the ends of thetubes, creating microchambers 35. Changes in fluorescence or in opticalabsorbance 36 may be detected in each tubular element through thetransparent end windows, and the reaction followed calorimetrically orfluorometrically.

[0089] A variety of other reactions may be performed inside themicroarray or inside the hollow fiber used to prepare a microarray. Forexample, a polypeptide, polysaccharide or polynucleotide may besynthesized in situ and/or a library of combinatorial small moleculessuch as esters, amides, carboxylates etc., prepared. The same reactions,including PCR, may be performed in any of the other types of fibers,including solid fibers if the fibers are sufficiently permeable to thereactants.

[0090] In one embodiment, an array device is envisaged comprising asolid phase and impermeable hollow fibers, where the hollow fibers forma multiple channeled surface. Such a surface can comprise multipleand/or groups of channels where the channels are distinguishable by, butnot limited to, differences in channel composition. Further, thechannels can extend to at least two exterior surfaces of the solidphase. Moreover, the solid phase may be bound to or integral with arigid solid support. In a preferred embodiment, the rigid supportcomprises wells for delivering fluids to subsets of channels comprisingthe solid phase. In a related aspect, the channels comprise immobilizedreagents that may be identical for each channel or group of channels ornon-identical. In one embodiment, the channels have a range in diameterof between about 1 μm to about 3 μm. In another embodiment, the channelshave a range in diameter of between about 0.45 μm to about 5 μm. In apreferred embodiment, the channels have a range in diameter of betweenabout 0.05 μm to about 8 μm. In a more preferred embodiment, thechannels have a range in diameter of between about 0.033 μm to about 10μm.

[0091] Further, such a device having discrete channels can have varyingcross-sectional areas. In one embodiment, the cross-sectional area ofthe channels has a range of between about 1×10⁻² μm² to about 10 μm². Inanother embodiment, the cross-sectional area of the channels has a rangeof between about 1×10⁻³ μm² to about 30 μm². In a preferred embodiment,the cross-sectional area of the channels has a range of between about5×10⁻³ μm² to about 60 μm². In a more preferred embodiment, thecross-sectional area of the channels has a range of between about 8.5×10⁻⁴ μm² to about 80 μm².

[0092] In a related aspect, the channels can also vary in inner surfacearea. In a preferred embodiment, the inner surface area of the channelsis from about 100 to about 1000 times the cross-sectional area of thegroup of channels. In one embodiment, the inner surface area of thechannels has a range of between about 100 μm² to about 1×10³ μm². Inanother embodiment, the inner surface area of the channels has a rangeof between about 50 μm² to about 5×10³ μm². In a preferred embodiment,the inner surface of the channels has a range of between about 10 μm² toabout3×10⁴ μm².

[0093] In another related aspect, the groups of channels can havevarying areas on the exterior surface of the device. For example, in oneembodiment, the channels have areas in the range of between about 2×10³μm² to about 3×10⁴ μm². In another embodiment, the channels have areasin the range of between about 200 μm2 to about 1×10⁵ μm². In a preferredembodiment, the channels have a range of between about 100 μm² to about3×10⁵ μm². In a more preferred embodiment, the channels have a range ofbetween about 20 μm to about 3×10⁶ μm².

[0094] In a further related aspect, the channels can vary in the numberof channels per cm² of solid phase surface. For example, in oneembodiment, the number of channels per cm² can be between about 600 toabout 700. In another embodiment, the number of channels per cm² can bebetween about 500 to 1000. In a preferred embodiment, the number ofchannels per cm² can be between about 450 to about 2000. In a morepreferred embodiment, the number of channels per cm² can be betweenabout 400 to about 4400.

[0095] When hollow, the microarray may have no agent of interestimmobilized thereon or therein. In such a situation, one has a verysmall multiwell plate, a commercial product per se. By placing, with orwithout immobilization, biological cells in “empty” hollow fibers; onecan use the microarray to determine the cellular response to a specificagent. One may even coimmobilize a substrate or reagent with thebiological cells to stimulate production of a detectable product whencontacted to or to interact with a specific analyte.

[0096] For both measuring the effects of a reagent on biologicalcellular material and a compound, one may coimmobilize the reagent inthe same fiber. Particularly preferred is to have the immobilized agentof interest and reagent(s) in a coaxial relationship within the samefiber. The solidifying matrix holding the reagent(s) may be dissolvable,meltable, degradable, or reversible (e.g. alginate with calcium ions orsodium EDTA) to further enhance interaction. As an example, part of afiber may contain an immobilized antibody in an insoluble polymer suchas a polyurethane. In another part of the same fiber, a labeled antigenis held in a water-soluble matrix such as 7% sodium stearate. Uponadding a target sample, potentially containing unlabeled antigens inwater, to a sliced bundle microarray of the fiber, the unlabeled targetantigens compete for the immobilized antibody with labeled antigens oncethe soluble matrix has dissolved. Because the microarrays of the presentinvention are very thin, dissolving of the soluble matrix and free ofthe reagents is very quick.

[0097] Coextrusion and injection of one material inside the mass of asolid are two other preferred methods for ensuring close contact betweenthe reagents and agents of interest.

[0098] As an alternative method for arranging to have reagent in closecontact to the agent of interest, two different bundle sections areformed. The first consists of immobilized agents of interest. The secondconsists of corresponding reagents entrapped in a soluble matrix. Onesection of each are aligned and in contact with each other such that theend of one fiber contacts the end of another fiber. By adhering the twosections to a solid phase, one on top of the other, the dissolving ofthe soluble matrix necessarily causes the reagent to contact theimmobilized reagent of interest. This contact is enhanced by usingimpermeable walled tubing to make the fibers and by sandwiching thereagent section between an impermeable solid phase and the section withimmobilized agent of interest. In such an arrangement, the only way forthe reagent to diffuse out of the microarray is to pass through the cellwith immobilized agent of interest.

[0099] While the usual technique is to place the molecules or biologicalcomponents inside the fiber before it is cut to form a microarray, it isan embodiment of the instant invention to place the molecules orbiological cells inside the hollow fiber after the microarray is formed.One example is the use of such a microarray to clone biological cells,viruses or other particles by adding a dilute suspension to themicroarray. Adding many individual agents of interest may be tedious butis an acceptable use. To compensate for potential spillover intoadjacent array cells, one may simply leave one or more rows of emptycells between each array cell being “filled” with an agent of interest.

[0100] The inside surface of the small tube described may be modifiedchemically to allow attachment of polynucleotides, polypeptides,polysaccharides or other molecules either directly or through linkers.The molecules attach, thus increasing the number of reactive sitesinside the tube. Since DNA and RNA are conventionally synthesized onsmall polystyrene beads, the most direct approach to a nucleic acidarray is to synthesize oligonucleotides on small polystyrene beads, withdifferent batches of beads having different sequences attached, and thento fill small polyethylene, polypropylene, polystyrene or other plastic,metal or ceramic tubes with the beads, packing down to completely fillthe tubes. The beads may be kept in place by careful heating thereof tosinter same or residual latex is added to the tubes and dried in placewith air pumped through the tube.

[0101] In a related aspect, agents of interest can be attached to innersurfaces, such as, but not limited to, hollow fiber channels, by firstderivatizing such agents using terminal primary amine groups andreacting the modified agents with an epoxysilane derivatized innersurface. For example, oligonucleotide probes can be attached to channelsurfaces through primary amine groups incorporated into the probe priorto immobilization. Such derivatized probes are then reacted withepoxysilane present on the channel surface, which results inimmobilization of the probe.

[0102] A seventh class of tubes or fibers includes tubules withpermeable walls. Methods and procedures for producing hollow selectivelypermeable fibers for use in kidney dialysis machines and for molecularweight fractionation have been developed (U.S. Pat. No. 4,289,623, U.S.Pat. No. 3,976,576) and are in wide current use. Procedures forembedding such fibers in solid sectionable plastics also have beendeveloped and are used to attach the fibers to tubing at the dialyzerends.

[0103] Permeable hollow fibers may be used in the instant invention intwo ways. In the first, the fibers are filled with reactant-carryinggels while already embedded in plastic. By carefully splaying out thefibers going into the cast portion, each tube can be filled selectivelyas previously described. That technique offers the advantage ofproducing small arrays quickly, and of developing new assays withouthaving to go through all of the steps required to produce separatehollow fibers, fill same with reactants, arrange same in arrays andinfiltrate same with the supporting plastic.

[0104] The second method of use involves filling the hollow fibersbefore being embedded in plastic. Techniques have been developed forcontrolling the wall permeability of permeable tubes. That allows theinflux and outflux of monomers and gelling agents during gelation to becontrolled, and for dialyzable agents to be removed after gelling. Forexample, acrylamide gels may be produced from acrylamide andbisacrylamide by cross-linking with ultraviolet light in the presence ofriboflavin. That technique is preferred when the specific bindingcomponent is heat sensitive or sensitive to other chemicals. Thecatalyst, which would interfere with subsequent fluorescencemeasurements, can be removed by dialysis through the tubing wall afterpolymerization.

[0105] Another gelling material is an isocyanate-containing prepolymerthat polymerizes on contact with water and generates only carbon dioxideas a byproduct of polymerization. The binding component may beincorporated onto solid phase(s) first or otherwise placed in the fiber,which then is polymerized and/or dried to incorporate the bindingcomponent to be used on hydration of the gel.

[0106] Permeable supporting tubing also allows the gel inside a tube tobe infiltrated with substances that render the reactants more stable,increase the physical strength of the gel and facilitate sectioning. Forexample, sugars such as lactose, trehalose, glycerol, fructose and otherpolyhydric alcohols may be introduced to stabilize proteins and to addsolids to the gels to assist in sectioning. The additives may be removedpartially from the exposed surface of the chip during use to make buriedreactive groups available. Additives diffusing into the gels also may beused to increase the strength and volume of a gel after it has beendried.

[0107] Also, when a particle containing the ligand or receptor isembedded in a fiber, the embedding medium may be soluble or meltable soas to be removable after the microarray is formed. By removing theembedding medium, more active sites on the particle are exposed forbinding. That variation is suitable when the particle is actuallymicrofibers or microbrushes of microfilaments having the immobilizedligands or receptors thereon similar to the cross-linked polymers of(17) in FIG. 3.

[0108] Once the tubes are filled with the respective gels and reagents,the outside of the tubes is cleaned, may be treated with reagents toincrease the adherence of the infiltrating supporting plastic and thenbundled to produce the product for sectioning.

[0109] An eighth class of tubes or fibers includes those synthesized bycleaving from a larger block, preferably a disk. The fiber materialcontaining the molecule of interest first is cast as a disk and then along fiber is peeled from the circumference of a rotating disk. Thattechnology is essentially the same as a smaller version of producingwood veneers where the veneer is peeled from a rotating log. Thetechnique has certain space and handling advantages over a long thinfiber. Such a disk also is more easily stored, particularly when activecomponents therein require maintenance under certain conditions, e.g.freezing, submergence in buffer, in the dark etc.

[0110] Arrays or parallel fibers may be attached together by manytechniques. A preferred one is by vapor sintering. The vapor, perhaps ahot solvent, is allowed to interact with the array for a specifiedperiod of time and then is removed by evacuation. In heat sintering, thearray is placed under lateral compression and the array heated to thesoftening point of the plastic. Another means is the use of low meltingpoint metals, such as gallium. By low melting point is meanttemperatures at or about physiologic temperature of the bindingcomponent.

[0111] A variety of histological embedding media has been developed thatpreserves biological molecules in reactive form. For example, Durcupan,Nanoplast and Quetrol 651 can be cured by very mild heating; JB-4 andImmunobed can be polymerized at room temperature; and the water solubleacrylic polymers, London Resin Gold and Lowicryl, polymerize at belowfreezing temperatures by ultraviolet light (all are available fromPolysciences Inc.). Conventional embedding media use solvents and waxes,and the waxes must be at least partially removed before analysis.

[0112] Optionally, use of materials that change the physical dimensionsof the fibers or chips under various conditions is also envisaged. Inone aspect, the use of such materials can afford the use of largermaterials that can be made smaller. For example, such a material wouldinclude, but not limited to, heat shrink plastics. A fiber made of sucha material could be used to make sections made smaller by heating beforeor after attachment to a solid phase. In one embodiment, the sliced chipmay be made as a “macro” array then shrunk down to a “micro” array. Inanother embodiment, once heat shrunk (or chemically shrunk as dried or ahydrophilic gel put in a hydrophobic organic solution or vice versa orstrong ionic strength solution etc.), the slice may be thicker but havea smaller diameter. In a related aspect, because some of thetemperatures required to heat shrink some plastics may denature someproteins, the invention may be used for, but not limited to, microarraysof peptides, oligonucleotides, DNA and combinatorial compounds. In a oneembodiment, slow heating is used so that the sliced chip does notcrumple into a ball. In a preferred embodiment, the heat shrink plastichas good shrink properties at low temperature (for example, see Paleariet al., U.S. Pat. No. 6,063,417).

[0113] In another embodiment, the sliced chips are prepared fromdehydrated or shrunken fibers. When such dehydrated or shrunken slicesare placed in water (or other solvents), the slice absorbs the water andexpands. In a related embodiment, the material surrounding the fiber orpart of the fiber is unaffected and the swollen material will protrudemaking a raised pad. For example, a polyacrylamide gel may be surroundedby opaque polypropylene (carbon black pigment) and the clearpolyacrylamide may form a curved surface. In a preferred embodiment sucha surface may act as a lens for, but not limited to, fluorescence, lightscattering, chemo- or electroluminescence, color formation and staindetection. In a related aspect, to control the amount of dryness to adesirable level for storage, the sliced chip may be placed in a moisturetight container, perhaps with a desiccant. Alternatively, glycerol,humectants or lubricant may be added so that the sliced chip willmaintain flexibility. Such substances may be removed before use.

[0114] In an alternate embodiment, porous chips are placed on a poroussolid support. In a preferred embodiment, agents of interest or bindingpartners are forced through the porous array by flow, resulting indiffusion distances in the flow through array in the nm range, reducingrate limiting diffusion/hybridization incubation times.

[0115] Embedding and sectioning methods therefore are available toidentify and localize specific biological molecules. In the case ofnucleic acids, specific nucleic acid targets can be detected by, forexample, in situ hybridization and amplification of specific sequencesby the polymerase chain reaction (PCR) and other nucleic acidamplification techniques (LCR, RCA, SDA etc).

[0116] The method of embedding is one that preserves the desiredcharacteristic or characteristics of the binding component in abiological cell. Thus, if antibodies were immobilized in a cell and itis the antigen-binding specificity of the antibody that is desired, theimmobilization method will be one which retains the antigen-bindingability of the antibodies. The method and means of attaching the fibersto form the array are also ones, which retain the antigen-bindingability of the antibodies.

[0117] Similarly, if the cells contain candidate molecules for bindingto a hormone receptor, the immobilizing and attaching method and meansare those that retain the configuration of the candidate molecules thatallows recognition and binding by the hormone receptor.

[0118] In addition, many protein or carbohydrate antigens may bedetected using immunological reagents. Detection is generally byincorporation of a fluorescent dye into the analyte or into the secondlayer of a sandwich assay, or by coupling an enzyme to an analyte or asecond or third layer of a sandwich assay that produces an insolubledye, which may be fluorescent.

[0119] Some solid phase surfaces may be used directly to immobilizereactants; others must be modified to allow such additions. Antibodieswill adhere to clean polystyrene surfaces, as will many other proteins(Van Oss, C. J. , & Singer, J. M. The binding of immune globulins andother proteins by polystyrene latex particles. J. ReticuloendothelialSociety 3: 29040, 1966.) Polystyrene, either in the form of microtiterplates or beads, has been modified to bind biological molecules, suchas, polynucleotides, polypeptides and polysaccharides. Perfluorocarbon(such as fluorocarbon polymers known as Teflon®), includingpolytetrafluoroethylene (PTFE), polyvinylfluoride, polyvinylidenedifluoride and perfluorodecalin, surfaces bind proteins or otherbiological molecules (U.S. Pat. No. 5,270,193). Such surfaces can bemade to include fluorinated surfactants, which may render the surfacehydrophilic, or positively or negatively charged. Glass, includingcontrolled pore glass, may be modified to allow covalent attachment ofantibodies, antigens, polysaccharides, polynucleotides, nucleic acidsand the like. Plastic surfaces may be modified non-specifically usingcorona plasma discharge or electron beam radiation and then may becoated with a variety of coatings or adhesives to which macromoleculesmay be attached. More specific covalent attachment of biologicalmolecules may be achieved by a variety of modifications, which attachreactive groups to polystyrene, or acrylic surfaces, which groups, withor without extending linkers, then will couple under mild conditions tothe biopolymers. In a related aspect, the solid phase may be made fromglass or silicone, including, but not limited to, nanochannel glass andoriented array microporous silicon.

[0120] A variety of chromatographic media also has been adapted tosupport immobilized bioreactants. Such media include soft gel beads,generally composed of acrylamide, agarose, Sepharose, which may bechemically cross-linked, and less compressible beads designed forhigh-pressure chromatography. A natural product useful as animmobilization support is cellulose, which is readily available inpowdered form. The supports may be modified chemically to allow covalentbioreactant attachment, or may be purchased in modified form ready forattachment.

[0121] Long DNA or RNA molecules may be immobilized by being polymerizedin a gel and are retained purely by physical entanglement. An example isthe retention of DNA in agar or acrylamide gels. In addition, otherbiological molecules, such as polypeptides, proteins, polysaccharides ornucleic acids may be linked covalently to long polymers so that, whenembedded in a gel, diffusion does not occur and the biological moleculeremains available for reaction with soluble reactants. Examples includethe attachment of proteins or nucleic acids to polyethylene glycol(so-called PEGylation) or to linear acrylamide chains.

[0122] In addition to methods by which a receptor or molecule ofinterest is immobilized and used to bind an analyte, general methodsexist for immobilizing members of a class of reactants. For example,protein A or protein G may be immobilized and used subsequently to bindspecific immunoglobulins, which in turn will bind specific analytes. Amore general approach is built around the strong and specific reactionbetween other ligands and receptors such as avidin and biotin. Avidinmay be immobilized on a solid support or attached to a gel and used tobind antibodies or other reactants to which biotin has been linkedcovalently. That allows the production of surfaces to which a variety ofreactants can be attached readily and quickly (see Savage et al.,Avidin-Biotin Chemist: A Handbook. Pierce Chemical Company, 1992).

[0123] A wide variety of methods has been developed to detect reactionsbetween immobilized molecules of interest and soluble reactants. Themethods differ chiefly in the mechanism employed to produce a signal andin the number of different reagents that must be sandwiched togetherdirectly or indirectly to produce that signal. Examples includefluorescence (including delayed fluorescence) with the fluorescent tagcovalently attached to the analyte, fluorescence involving soluble dyes,which bind to an analyte, and similar dyes wherein the fluorescencethereof greatly increases after binding an analyte. The latter can beused to detect nucleic acids. In more complex systems, includingso-called sandwich assays, the result is the immobilization in thedetection complex of an enzyme that, in combination with a solublesubstrate, produces a preferably insoluble dye that may be fluorescent.Alternatively, the detection complex attached to the bound analyte mayinclude a dendritic molecule, including branching DNA, to which isattached many fluorescent dye molecules.

[0124] In a related aspect, there are fluorescent dyes that binddirectly to agents of interest. For example, rare earth metal chelatescan be used such as, but not limited to, holmium, europium, terbium,samarium, ytterbium, neodymium and dysprosium. In a preferredembodiment, the rare earth metal is europium. In a further relatedaspect, heavy metals such as, but not limited to ruthenium can be used.Such dyes are available commercially from, for example, MolecularProbes, Inc. (i.e., SYPRO® Ruby Protein gel stain and SYPRO® RoseProtein blot stain).

[0125] Methods for making dental floss having attached short transversefibers to give a brush-like configuration may be modified to allowattachment of reactants. Patterns encoding identifying information onstrands or fibers may be employed in the form of small linearly arrangeddots. In the development of multifiber endoscopy arrays, methods forchecking the array have been developed in which a light beam or rasterimage is introduced at one end of the fiber bundle in such a manner thatthe light sequentially illuminates each fiber. The pattern of emittedlight exiting the other end then is determined. If identical, no fiberis out of place.

[0126] The art of detecting bubbles or voids in liquid filled tubing isknown and may depend on differences in refraction, light absorption orfluorescence as measured along individual tubes.

[0127] The art of using centrifugal force to fill short lengths oftubing with viscous media is evident to those trained in the arts.

[0128] Other methods include the use of microforges and automaticnanoliter injectors. For example, a microforge can produce glassmicropipet tips with sizes of 10-30 1 μm. In one aspect, the capillaryglass is produced from N-51-A material and has a softening point of 780°C. Tips can be pulled using a microforge (e.g., TPI Microforge athttp//www.techproint.com/microforge.htm/) and broken with forceps. Suchtips can then be backfilled with oil (or other non-compressible fluid)and attached to an automatic nanoliter injector (e.g., Drummond“Nanoject II,” Drummond Scientific Company Broomall, Pa. ). Using such adevice, a fully extended plunger can reproducibly withdraw substantiallyabout 5 μl of fluid. In a related, aspect, viscous samples may bewithdrawn in small steps allowing the sample to equilibrate in the tipbefore continued filling. Fibers made from such tips are amenable tosectioning by microtome, after which, in the case of hollow fibers, forexample, the capillary tube cladding can be removed.

[0129] Microtomes for sectioning tissue blocks which may contain samplesranging from soft tissues to bone, often in blocks of embedding material(e.g. wax), are commercially available, as are a variety of techniquesand arrangements for attaching sections to glass or plastic slides, fortreating the slide automatically to remove some or all of the embeddingmedia, and for systematically exposing the slides to a series ofreagents.

[0130] Microtomes and other sectioning or cutting instruments capable ofcutting assembled bundles of tubes into thin sections, and ofmaintaining the orientation of the component tubes after sectioning areknown. Blade cutting may reduce contamination of binding componentsbetween cells of the microarray.

[0131] The microarrays can be of any thickness as required by theanticipated use thereof. Another determining factor might be therigidity of the fiber bundles. It is likely the sections will be lessthan 1 cm in thickness. It is likely the sections will be less than 50mm in thickness. In one embodiment, the thickness of the sliced fiber(or block) is between about 100 μm and about 1000 μm. In a preferredaspect, the thickness of the sliced fiber is less than about 50 μm. In amore preferred embodiment, the thickness of the sliced fiber is lessthan about 20 μm, as will be exemplified in further detail hereinbelow,sections can be on the order of microns in thickness.

[0132] The sections (as microarray chips) may be attached directly toadhesive surfaces on flexible films or on solid surfaces, such as glassslides. It is also feasible to attach sections (the word “section” isused here in place of “chip”) at intervals along a film strip, withothers interleaved therebetween. Thus, a set of about a dozen or moresections that are different may be placed in repeating order along thefilm, and the film then cut to give one set. For sequencing studies, oneDNA insert may be amplified, labeled, and the hybridization patternthereof to a large set of sections examined.

[0133] In one embodiment, the reagent can be incorporated into a pasteor a gel that remains firm or quickly hardens after being extruded ontoa solid phase. This is a variation of the conventional spottingtechniques. Materials include, but are not limited to drying oils,conventional paint materials, molten materials which dry or cool to thepoint of becoming very solid, photo-, UV- or heat polymerizing orcrosslinked materials and corresponding deposition treatment. Forexample, in one embodiment, coating a slide with calcium chloride andadding a thickened suspension comprising alginate, which furthersolidifies as the calcium ions dissolve and diffuse through the paste orgel is envisaged. In a related aspect, the binding partner and matrix asa core material are co-extruded as a coaxial outer coating.

[0134] By using a non-deformable bundle of fibers, one can cut or sawthe bundle transversely thereby forming a large number of identicalplates that are perfectly realignable. That permits highly consistentand reproducible arrays. By using an easily detectable differentmaterial for one or more fibers, as a means for registering themicroarray alignment, realignment can be facilitated.

[0135] Most immunochemical or competition assays depend on a signalproduced by a reagent other than the analyte. However, methods forfluorescently labeling antigens, such as proteins containing aliphaticamino groups in a complex mixture have been developed which arereproducible and quantitative. For example, CyDyes supplied by AmershamLife Sciences, and particularly, Cy2, Cy3 and Cy5 are useful. When thecomponents of such labeled mixtures are reacted with an array ofimmobilized antibodies, each specific antibody binds to one of thefluorescently labeled analytes, and the presence of each of thespecifically bound labeled analyte can be detected by fluorescence. Thatmethod can be improved further by exposing the bound antibody array to asolution containing known subsaturating quantities of each analyteprotein in a non-fluorescent form, washing the array, and exposing thearray to a test mixture of labeled proteins, thus producing a multiplecompetition assay.

[0136] Any of the conventional binding assay formats involving animmobilized binding partner may be used with the microarray systems ofthe instant invention. Briefly, the microarray may have either pluralligands or plural receptors and the analyte may be either plural ligandsor plural receptors. Competing elements that bind to either the analytesor the microarray cells may be added. The sample may be labeled and/orthe competing element may be labeled and/or the microarray cell may belabeled. The labels may be interacting with each other to make adetectable signal or product, or to quench a signal or product. Thenumber of different combinations is in the dozens and any may be used inthe instant invention as well as different combinations for differentcells of the microarray assay.

[0137] Often several different clinical tests are required to define aparticular disease. The multiple tests often are done serially, with onetest or member of a battery of tests suggesting another, which in turnsuggests a third test or group of tests, some of which are rarely donein local laboratories. There is therefore a need for inexpensive chipsfor the performance of a series of branching batteries of testsconducted simultaneously, using methods that produce accurate numericalresults in a machine readable form, which are stable over time, andwhich are read by devices that can be compact and inexpensive relativeto currently clinical analytical systems.

[0138] The availability of inexpensive microarrays testing for manydisease markers simultaneously may provide indications of the severityof the disease and/or its prognosis. The diagnosis and subcategorizationof each diagnosis is further enhanced in the present invention bymeasuring combinations of markers. Additionally, it may unexpectedly bediscovered that the patient sample actually has a second disease presentor that the disease may involve an additional organ system. Also, theoverall health of the patient may simultaneously be measured. Whenperforming one test at a time, as in the prior art, one must firstsuspect an abnormality in order to request a test for it. With thepresent invention, it is equally easy to measure 1 marker as 500markers, which would lead to a qualitative difference in how diseasesare diagnosed.

[0139] Many biochemical analyses require that the analytical procedurehave wide dynamic range. Thus, enzyme and immunochemical assays oftenare done by determining the course of a reaction over a period of time,or by doing multiples analyses on a series of dilutions. Such analysesmay be done by “reading” the microarrays at intervals during exposure toan analyte mixture of a developing reagent. In addition, parallelanalyses using standards and blanks (controls) are required and areincluded. Large numbers of standardized inexpensive biochips will berequired to meet such needs. The biochips may incorporate reactants ofdifferent classes that can, for example, detect and measure antigens,drugs, nucleic acids or other analytes.

[0140] In one embodiment, a microarray comprises a solid phase having aplurality of structures bound to its surface. In a related aspect, thestructures comprise an immobilized agent of interest that is availablefor binding to a target and such a microarray can contain a plurality ofdifferent agents of interest in a corresponding plurality of differentstructures. In a preferred embodiment, the structures are permeable tothe target and the structures comprise a material that includes, but isnot limited to, plastics, gels, glass, sols, colloid suspensions anddextrans. Further, the structures may have hollow inner surfaces. Inanother embodiment, the structures are impermeable to the target and thetarget binds to the exterior boundaries of the structure.

[0141] These structures have three-dimensional form and are more than asingle layer of molecules bound on the microarray solid support. Theseare either formed or carved out from a larger structure.

[0142] The structures in this embodiment may comprise the solidifiedcontents of a tubular fiber where the outer tube has been degraded orremoved leaving behind a microarray having structures resembling smallpillars with a gap between them where the tubular material once was. Thedegradation or removal of the outer tubing may be accomplished bydissolving with a solvent, melting or subliming with heat or chemicallydegrading. Physical removal of the outer tubing where the solidifiedcontents remain adhered to the solid support may be done by having thesolidified contents (but not the tubing) bound to the solid support byphysical cleavage with a knife or similar instrument, or by laser,electrical arc or other electromagnetic irradiation to destroy andthereby remove whatever material present between the pillars whichremain.

[0143] The solid support may be precoated with a material, which willadhere the solidified contents but not the tubing, thus permittingeasier removal. Also, the material may selectively adhere particles andtiny structures in the sliced fiber section without adhering thesolidifying matrix.

[0144] Other techniques for producing a three dimensional structureinclude depositing a three dimensional structure directly on the solidsupport surface. This may involve a preformed structure or a fluid orsemi-fluid material which solidifies very quickly before it spreadssignificantly or forms a few molecule thick layer. The preformedstructure may be a long fiber that is cleaved once it adheres to thesolid surface thereby depositing the structure. An analogy to the latermethod is seen in the food art. Chocolate chip cookie dough issemi-solid at the time it is extruded or deposited on the cookie sheetwith the dough representing the matrix and the chocolate chipsrepresenting the agent of interest. Alternatively, a larger piece ofmaterial may be cut and pieces deposited such as the process shown inFIG. 8 without having the individual cubes (or other shapes) adhered toeach other.

[0145] By coextruding two different materials, one may form a coaxialstructure where the outer layer may be impermeable plastic to form amicrowell or separate the inner layer having the agent of interest fromother coaxial structures having a different agent of interest. Cleavingthe top of an extruded or deposited structure to yield a smooth surfacefor contacting the target is frequently desirable for a number ofdifferent three dimensional structures made by a number of differenttechniques.

[0146] Arrays have numerous uses other than determining bioactiveproperties. Chemical interactions and reactions may be tested as well.Such an assay can, for example, enable testing different reactivechemicals simultaneously against a test substance or material todetermine corrosion, electrochemical reaction or other interaction. Thatis particularly advantageous in the chemical formulations of pluralsubstances such as in cosmetics, paints, lubricants etc. Alternatively,one may assay for desirable interactions between the analyte and all ofthe molecules of interest in the array.

[0147] The microarray of the present invention has many othernon-microarray uses such as using the resulting surface for affinitychromatography, affinity separations, protein-protein binding to formprotein complexes and the measurement for all of these.

[0148] The present invention also may also coat the surface withmaterials other than organic chemicals and biological materials.Different metals, anticorrosive coatings, decorative or instructionalcoatings, coatings for surface plasmon resonance (see U.S. Pat. No.5,955,729), coatings for SELDI (see U.S. Pat. No. 6,020,208),combinatorial libraries of chemicals, and even coatings for depositingphotoresists, electrically conductive coatings etc. such as are used inelectronic integrated circuits.

[0149] A general problem with use of gels for the immobilization ofreactants has been that reactants, which may attach to gel-immobilizedagents of interest, require considerable time to diffluse into and outof the gel. Where the detection is by fluorescence, inclusion of a dyeabsorbing the excited light into the gel limits detection to a regionclose to the surface. Inclusion of the ultraviolet light absorbingmonomer, 4-methacryloxy-2-hydroxybenzophenone (Polysciences, Inc.) in anacrylic embedding medium can solve the problem. Addition of a quenchingmolecule such as DABSYL or DABCYL to accept the vibrating excitedmoieties before fluorescence emission also may be of use.

[0150] When one wishes to enhance binding between analyte and bindingpartners on the surface area of particles in a fiber of the microarray,one may etch the embedding matrix of each fiber, thereby exposing moreof the surface area of particles in each fiber of the microarray.

[0151] When performing a binding assay, one may wish to encouragediffusion of the analyte into the microarray cell to increaseligand/receptor binding (sensitivity), to make the microarray morequantitatively reproducible and to enhance spectorphotometric detectionif done by passing light through the microarray. To enhance diffusionthrough the microarray, one may force the ligand through the microarraygel material. That may be done by mounting the microarray on a porousmembrane and passing the ligand and or ligand solution through themicroarray by hydrodynamic, electrophoretic or mechanical means. Forexample, fluid may be flowed through the microarray by pressuredifference on each side of the membrane. Fluid also may be drawn throughby simply applying a stack of paper towels on the backside of themembrane to draw fluid through the microarray. As for electrophoreticmeans, a potential is applied across the microarray either across theentire microarray or using single point electrodes located on both sidesof a single or group of cells of the microarray. Mechanical means mayinvolve a pump of various configurations to mechanically push or pullfluid through the microarray by providing a pressure differential.

[0152] Using a porous membrane also has certain advantages in washingthe microarray to achieve lower backgrounds. If porous particles orthreadlike components are embedded within the fiber, sectioning throughthe porous particle or threadlike component may make the resultingstructure more porous and allow greater surface area contact to bothreagents and washing. Etching of an embedding medium or capillary alsoincreases porosity and exposure to the immobilized molecules ofinterest.

[0153] If a porous particle is sectioned, preferably twice, largerchannels allowing passage that is more fluid may be present. Fibers withsectioned particles may be mounted over permeable membrane supports orover holes in a solid base support. The result allows fluid to passthrough the cells of the microarray.

[0154] By using the instant invention, one avoids the difficulties ofindividually spotting each cell on a solid phase or forming a compoundat each cell. The former method is limited by human intervention andapparatus, as well as the ability to measure quantitatively smallamounts of liquid. The latter technique is limited by the types ofcompounds that can be synthesized on the solid phase. Both prior arttechniques are expensive and require elaborate automated equipment ortedious labor as each array is produced individually. By contrast, theinstant invention is technically simple and quick where the “batch” isin the thousands to millions of microarrays. The only individual effortrequired for each microarray is the step of cutting.

[0155] Microarrays prepared from sets of stored reagents or by thesynthesis of different reactive sequences or compounds on the base chippresent difficult problems in quality control. With large arrays, eachreagent in final form cannot be separately assayed before being used,nor can the correctness of the in situ synthesized sequences be assureduntil after a set of arrays has been manufactured. If errors orsubstandard components are discovered in a batch of arrays, all must bediscarded. Those problems limit the use of “biochips” in routineclinical studies.

[0156] It is known that immobilized proteins and nucleic acids are morestable, especially in a dry state than in solution.

[0157] The agent of interest in the instant invention may comprise avery broad range of chemicals, complexes, biological cells or fractionsthereof. Nucleic acids, many proteins, proteins which have been modifiedor are coated with detergents such as sodium dodecyl sulfate are solublein organic solvents and a wide range of organic compounds and thus canbe incorporated into polymerizing mixtures such as those used to produceplastics. Hence, it is technically feasible to produce long fibers ofacrylic or other plastics each containing a different agent of interestusing currently available extrusion technology for practice in theinstant invention.

[0158] Large numbers of different and potentially new active compoundsmay be screened simultaneously by immobilization in fibers, bundling,sectioning and. forming a microarray. Peak fractions from separations,such as plant extracts, may be collected simultaneously and used to forma microarray. The microarrays then may be used in a large number ofassay systems simultaneously, dramatically reducing the time and effortto screen all of the compounds present for whatever activity onechooses.

[0159] Particularly preferred are large numbers of proteins or peptidesgenerated by mass techniques. Different fractions from a separationtechnique from a natural source provide a resource of many differentproteins and peptides. A number of fractionation procedures are known toseparate mixtures of many compounds. Different fractions or specificcompositions may be used to form a single fiber. Two dimensionalelectrophoresis gels from serum and other tissue and natural sourcesproduce thousands of different proteins separated on the gel. Each maybe removed individually (e.g. cut, eluted etc.) from the gel and used asthe molecule of interest to form a single fiber. In such a method, withdifferent bundles being formed from different samples, proteindifferences between different samples may be readily compared.

[0160] When the immobilized macromolecules are antibodies, themicroarray may be used to diagnose a variety of protein-based anomalies.A labeled second antibody to the protein of interest may be used tohighlight the cell further. In addition, the array may be used toimmobilize infectious agents, which have been either stained previouslyor which, are stained after immobilization. Thus, microbes frombiological samples, e.g. serum or plasma, may be concentrated, stainedwith a fluorescent nucleic acid stain, such as TOTO-1 or YOPRO-1, andthen allowed to find matching antibodies on the array. Then the boundanalyte may be detected by scanning for fluorescence and identified byposition.

[0161] It is equally a part of the instant invention to immobilizemicroorganisms or other molecules of interest and use the immobilizedreagent to localize antibodies from a fluid from an individual, and thendiscover the location of the latter using a fluorescent anti-humanantibody, thus diagnosing a disease which elicited antibody productionin the first place.

[0162] Arrays have been prepared using phage display'with inserts fromspecific genes, using synthetic oligonucleotides, or, to a limitedextent, using displayed antigens or antibodies. In the instantapplication, a population of peptide or antibody display phage may beused where each display phage is used to prepare a single fiber. In suchan arrangement, the phage is large enough so that some portion of eachsurface molecule will remain embedded in the gel or plastic, whileanother part will be exposed. The molecule of interest may be bound tothe fiber per se, entrapped inside the matrix or bound to a solid phaseparticle or tiny structure that is in or on the fiber. The phage,recombinant bacteria or other complex biostructure also may be fixed andthe contained proteins cross-linked using glutaraldehyde or similarfixative, if desirable.

[0163] Each fiber may contain a mixture of molecules of interest. Forexample, during chemical synthesis, a number of isomers are prepared. Itis convenient to not separate the isomers before forming a fiber in somecircumstances. Likewise, when fractionating a mixture, forming a fiberwith a mixture of receptors may be acceptable as total and completeisolation is difficult and time consuming.

[0164] When a collection of fibers are used, or in other embodimentswherein, for example, particles are embedded in a matrix to form afiber, a filling material to maintain the relative positioning of thefibers along the length of the bundle may be desirable. Various gluesand adhesives are known in the art. For example, a filling compositioncomprising an oil constituent with is a relatively high molecular weightaliphatic hydrocarbon of at least 600, an inorganic constituent and ablock copolymer thicken yet reduce the viscosity of the material. Anantioxidant also may be included. See, for example, U.S. Pat. No.5,187,763.

[0165] The filling material selected is one that maintains the fibers inregister, can be cut and does not interfere with any downstreamprocedures to which the microarray will be exposed. For example, othermaterials that can be used are polymerizable materials, such as apolyacrylamide.

[0166] The embedding matrix for the fibers may be black, opaque orotherwise adsorbent to emitted signals of a label to reduce cross talkbetween the cells in the chip. Additionally, any adhesive between thefibers may contain the same adsorbent material to reduce backgroundbetween cells of the microarray. Optionally, a specific layer of thematerial may be placed between the fibers before the bundle is formed.When hollow fibers are used, the opaque material may be incorporatedinto the hollow fiber shell itself.

[0167] Arrays may have an entire set of antigens/antibodies etc. in thevarious cells along with controls to screen blood samples for commonblood borne diseases before donated blood is provided for transftision.Likewise, certain symptoms have a number of common causes that may bescreened simultaneously for using arrays. For example, urinary tractinfections are common and may be caused by a large number of differentbacteria of varying sensitivity to various antibiotics. The simultaneoustesting for a number of different factors would save considerable timeand expense.

[0168] In the course of using a chip of the instant invention, variousknown techniques and materials are used to reduce non-specific reaction.Thus, in the case of a protein-based assay, the non-specific sites onthe chip contributed by the substance of the fiber or filament, theembedding material and essentially everything aside from the bindingcomponent of interest may be reacted with a blocking agent, such asalbumin or milk, so that the blocking agent will bind to those areas notcontaining the binding component which could react with a ligand,analyte, reporter molecule or whatever would specifically bind to thebinding component, as known in the art.

[0169] Arrays may have two or more identical cells made from differentfibers but containing identical binding agents. That provides aninternal quality assurance check for the array. Additionally, it ispreferred for some of the cells to provide different concentrations ofthe binding component for quantitative measurement of an analyte. Thoseprovide internal standards for the microarray for both qualitativedetection and quantitative detection. For example, a series of cells maycontain different concentrations of an antibiotic. When a samplemicroorganism is contacted with the cells and allowed to incubate, theabsence of growth in one cell and the presence of growth in another cellprovide an approximate minimal inhibitory concentration. The same can bedone for determining minimal bacteriocidal concentrations when stainedwith a vital dye such as trypan blue or fluorescein acetate. Since amicroarray may contain thousands of cells, one can determine theantibiotic sensitivity to numerous antibiotics simultaneously.Quantitative determination of other biological activities with eitherligand or receptor immobilized in the gel may be used.

[0170] Essentially the same fiber may be used multiple times in the samemicroarray. That provides an internal quality control check and improvesconfidence in the binding assay. That also provides additionalquantitative measurements if such an assay is performed to improveprecision. Blank fibers, fibers with no molecule of interest boundthereto, provide a good negative control and should be used in everymicroarray.

[0171] Long filaments, capillaries or coaxial two-material filaments arearranged in parallel and then sintered or adhesively bonded to formbundles which are preferably resistant to deformation, and in which eachstrand or capillary is continuous from one to the other. The positionalarrangement of fibers or capillaries should remain the same throughoutthe bundle. Filaments composed of two different types of material incoaxial formation may be used. The core material is made of a material,which can be dissolved, and the cladding being resistant to the samedissolving conditions. For example, strong alkali is capable ofdissolving certain types of glass but not others. The dissolving stepmay occur before or more preferably after sectioning depending on thematerials present.

[0172] Alternatively, the cladding may be dissolvable and the coreresistant leaving isolated “islands” on a microarray attached to abacking sheet. In either situation, the space left by the dissolvingstep may remain empty or be filled with a diverse material. Partialdissolving to yield a porous material is also part of the instantinvention. Porous materials have increased surface area, which isparticularly desirable for binding assays.

[0173] Particles, especially porous beads, may also be “chemicallysintered” to form a filament, sheet or inside of a capillary. Thattechnique also may be used to adhere different fibers together. One suchway is first to bind a molecule of interest to the particle. A blockingagent may be added to block any remaining active sites or adsorptionareas on the particle. If not already done, the beads are packed in atube or the hollow fiber. A chemically reactive compound whichcrosslinks or couples either the blocking agent and/or the molecule ofinterest and/or unreacted sites on the beads then is added and at thelocations where the beads touch, chemical adhesion results. The tube orhollow fiber may remain in place or be removed. The molecules ofinterest in the internal pores of the beads are not touching and thusare not altered significantly. Alternatively, the pores of the beads maybe filled with a hydrophilic solution and held by capillary action whilethe spaces between the beds are filled with a hydrophobic adhesive orsetting liquid.

[0174] A representative example of chemical sintering is to adsorbProtein G on porous beads and then to add a gelatin blocking agent. Theresulting beads are filled in a 1 mm plastic tube and then a proteincrosslinking agent added, e.g. carbodiimide. After the reaction iscomplete, unreacted reagents are washed free and then any suitableantibody of interest is added thereto to bind to Protein G, therebyforming a fiber suitable for bundling and cleaving to make a microarray.Alternatively, the surfaces of the particles may be biotinylated firstand avidin may be used as the crosslinking agent. One may use avidinlabeled antibodies instead of adsorbing Protein G to the beads. Anotheralternative is to use relatively large porous beads and an adhesive orembedding medium to fill the spaces between the beads. When the fiber issectioned, the beads are so large so as to be cleaved, thereby openingup the inside of the beads for the bound molecules of interest to beexposed. Hollow beads or microballoons may be used in lieu of porousbeads, as molecules of interest encapsulated therein will be exposed oncleavage of the bead. The concept is the same as sectioning a tissue orembedded cell to expose and visualize intracellular features.

[0175] Additionally, one may use two different sets of beads: set one isporous and has the receptor/receptor binding substance bound thereto,and the second set is coated with highly reactive material or modifiedwith a reactive group which will bind to the first set of beads orcoating thereon. A tube first is filled with both beads in dry form, thetube shaken and then fluid is pumped therethrough permitting a reactionto occur thereby forming a solid fiber of beads. Alternatively, if thefirst set of beads is quite large, the beads may be added first (with orwithout fluid) and the second set added later so that the beads filterdown through the spaces between the larger beads and react accordingly.The reaction between the beads may be through specific binding moietiesor of a non-specific binding reaction to form a crosslinking of thebeads into a sliceable solid. The second beads may be black to reducestray light in the fluorescence detection. Such fibers may or may notallow for flow after sintering depending on desired utility.

[0176] In one embodiment, fibers containing beads can be used for solidphase synthesis. For example, a nucleotide/amino acid/sugar/chemicalunit is first linked to a bead. The bead is preferably glass or apolymer, must be sinterable, yet allow the fiber to remain porous. Ahollow fiber is filled with the beads and the beads are sinteredtogether with heat, chemical, vapor, LW, solutions etc. In the situationof a polynucleotide binding partner in the fiber, one flows/pumps/usesnegative pressure/uses capillary action to percolate reagents throughthe fiber where the reagent reacts with the nucleotide to produce adinucleotide. After washing, the process is repeated until a sequenceproduct of desired length is generated. The resulting fibers can bebundled before starting synthesis with a random or patterned addition ofdifferent nucleotides to prepare many different binding partners in thefibers. For example, ¼ of each bundle is infused with T and appropriatesynthesis reagents, and ¼ each with G, A or C. The selection of whichfibers to infuse with which nucleotides changes with each round ofsynthesis.

[0177] In a preferred embodiment, one can make all possiblecombinations, where the final product is usable in methods including,but not limited to, DNA sequencing, probe synthesis and primersynthesis. In another embodiment, by binding to various sequences oftarget DNA in a sample, a pattern of DNA/RNA sequences in the sample maybe determined. Such a pattern may be a “fingerprint” for a particularabnormality, even in the absence of acquiring specific sequenceinformation beforehand. The measurement of many different types of MRNAto generate a sample “transcriptome” by other techniques is known in theart.

[0178] In a preferred embodiment, such in situ solid phase synthesis isequally applicable for generating any other “heteropolymer” for fiberimmobilization. In a related aspect, examples include, but are notlimited to, polypeptides, polysaccharides, large numbers of organicchemical monomers which are “mix matched” to generate combinatoriallibraries as well as polynucleotides. In a preferred embodiment, solidphase peptide synthesis is envisaged. Further, such solid phasesynthesis methods for polynucleotides and peptides are well known in theart (e.g., U.S. Pat. Nos. 4,816,513 and 4,965,349) and are readilyadaptable to bead-fiber immobilization as described in the presentinvention.

[0179] The solid phase particles inside a fiber may be chemicallysintered together after synthesis of the oligomer/polymer/heteropolymerin situ. If the solid phase particles are labeled with a differentspecific binding partner such as biotin, then a solution of acorresponding specific binding partner, such as avidin or streptavidinmay be pumped through the fiber and cause the particles to chemicallyadhere to each other.

[0180] After the fibers in the bundle are fused or otherwise adhered toeach other in a fixed pattern, the bundle is cut transversely or at anangle into many thin disks and portions are optionally dissolved ifdesired. When hollow capillaries are used, the resulting disks may beused as channel plates for the amplification of optical images and lightpipes. Regardless of whether rods or fibers are used, the thin disksalso may be used as filters because of uniform hole size.

[0181] Each fiber segment in the sectioned two-dimensional array wouldcontain relatively large numbers of binding components, such as DNA,RNA, or protein molecules. As a first step in the use of the finalarray, a solution, which can erode the plastic surface of the array veryslowly, is washed over the surface. That is done at a rate, which willremove any biopolymer molecules that become loose. The wash then iscontinued, grading into a solution that will not erode the plastic. Thearray then may be dried and stored until used, or may be used at once.To assist in exposing reactive agents of interest in the plastic,particles on the surface are dissolved, forming a solution and exposingthe molecules.

[0182] Because each fiber has the molecule of interest in the same formas will be present in the microarray, one can perform a quality controlcheck on the fiber itself rather than using the entire microarray. Thatis particularly important when the microarray is used for diagnosticpurposes. Sampling microarrays from a batch may be a quality controlcheck but it does not actually check the microarrays being sold. Bycontrast, small slices of the fibers themselves are being used in theinstant invention. Assaying the fiber itself represents an actual testof every microarray that has a slice of that fiber as a microarray cell.

[0183] By contrast, with solid phase in situ synthesis of a molecule ofinterest directly on each cell of the microarray, none of the actualcompositions to be used containing molecules of interest is actuallytested after synthesis. Rather, spot checking is relied on for qualityassurance. In microarray manufacture by spotting liquid droplets on asolid phase, one may test the liquids as a quality control check.However, the liquid samples do not represent the quality of the drymolecules of interest immobilized on a slide. Therefore, the qualitycontrol check is not the same as the actual product being sold. Again,one lacks any quality assurance for the actual compositions in the cellsof the microarrays being sold.

[0184] For quality control in the instant invention, the fibers may beindividually assayed, assayed in ribbons or small groups, or assayed aspart of the whole bundle before slicing. Furthermore, by testing onefinal microarray, one has effectively tested all of the microarrays asthe composition of the fiber is the same as that portion of the finalproduct.

[0185] The microarrays of the present invention have the ability to holdbiological cells or pieces of tissue at the individual addressablelocations of a microarray. This is particularly preferred with cellsthat can be suspended in a solidifying medium before being pumped into ahollow fiber. This finds particular application for leukemia or lymphomacells that are naturally suspensable. Microarrays so produced are partof the present invention. Bacteria and other microorganisms may also belikewise used to prepare microarrays for screening candidate compoundsfor antibiotic properties or specific binding properties, such as from aserum source.

[0186] For clinical tests, regulatory approval of tests and systems andmethods for making same is required. When chips are fabricated usingphotolithography and other technology derived from electronic chipmaking, the cost of individual chips is extraordinarily high, and thepossibility of error when chips are made individually is very high.Since chips are made individually and used only once, quality control isdifficult and there is no good way of proving that any given chip issatisfactory. The best that can be done is to test a large fraction of abatch at random. With the instant invention, a very large number ofsections can be made from one composite assembly, and adjacent sectionsintercompared as well as those some distance apart. Statistical analyseswill be able to predict the rate of errors that may occur. However, ofeven greater importance is the fact that since the sections can be madein large numbers and quite cheaply, it will be feasible to run duplicateanalysis on clinical samples, and to run confirmatory analysis whenimportant diagnostic results are obtained. The instant inventiontherefore makes feasible widespread and routine application of geneticanalyses in the practice of medicine.

[0187] The key agent of interest components of the fibers is retained bythe fiber by being immobilized therein. Immobilization may beaccomplished by a number of techniques, known per se, such as entrapmentin a matrix and chemical coupling, perhaps through a linking moietythrough an amino, hydroxy, sulfhydryl or carboxyl moiety. Chemicallyattaching the chemical to a monomer or being used as a monomer to bepolymerized also effectively incorporates the component. Binding alsomay be accomplished by a number of affinity techniques such as protein Aor protein G for antibody attachment, ligand/receptor pairs such asbiotin-avidin, HIV-CD4, sugar-lectin or through a ligand that has areceptor such as digoxigenin-antidigoxigenin. On the other hand, nospecific attachment is required for situations where a gel or a non-gel,gelling matrix, such as wax, silicone polymers and silicone emulsionsmay be used. Liquid wax or a gelling agent simply is mixed with the keycomponent and cooled to form a solid fiber by casting or extruding.

[0188] Arrays need not be assembled in a single step. Flat arraysconsisting of a set of tubes arranged side-by-side may be preparedfirst, and the end of the array sectioned and tested. The flat arraysthen can be attached together with a suitable adhesive to give athree-dimensional bundle. The use of intermediate flat arrays means thatthose can be prepared and stored, and custom two-dimensional arrays canbe prepared by selecting and attaching together differentone-dimensional arrays. The stepwise assembly procedure providesinspection at each step, minimizes losses due to errors or low bindingefficiency of one rod or tubule, and provides flexibility to assemblenew patterns of reactants.

[0189] For general clinical use, it is important to have identifiers onthe slide holding the chip, and identifiers may be integral with thechip itself. FIG. 5 illustrates chip 40 with array elements 41, and witha barcode 42 printed along one border to provide identification andorientation. In addition, small concentrations of dyes, usuallynon-fluorescent, may be incorporated into the polymers from made suchthat they present a pattern 43 to 44, for example, of one or morenumbers, or one or more letters. It is also useful to have a few cellsor elements which do incorporate fluorescent dyes and which serve tocalibrate the fluorescence measurements. It is further feasible tointroduce dyes into the contents of selected tubes to additionallyidentify them. Note that the diagonal line 43-44 further indicates thatthe horizontal rows of tubes from which the array is assembled, are inthe proper order. If tubes in an array are out of alignment giving riseto the loss of one tube or rod in one line, this can be readily observedbecause the entire pattern will show a misalignment.

[0190] The embedding material or adhesive used to hold the tubes in abundled configuration may be opaque, while the tubes and preferably thecontents thereof will conduct light along the entire length. As a finalcheck on the orientation of array elements, one element at a time at oneend of the bundle may be illuminated, and the light detected and relatedto array position at the other at the other end as shown in FIG. 6 wherebundle 50 with fibers 51 is illuminated by a cathode ray tube (CRT) 52generated raster 53 which is focused on the distal end of the bundle bylens 54, and the transmitted light recorded by CCD camera 55. Individualspots 56 yield signals 57 that are detected.

[0191] An arrangement for detection using epifluorescence is showndiagrammatically in FIG. 7 where chip 60 is illuminated by beam 61generated by lamp 62, which passes through filter 63 to isolate light ofa wavelength optimal for exciting fluorescence. A split-beam prism 64directs the exciting light toward chip 60. The emitted light passes backthrough the split-beam prism after which the emitted wavelengths areisolated by filter 65 and detected by CCD camera 66. Different systemsfor detecting fluorescence patterns on chips are known to those skilledin the arts.

[0192] As an alternative method to forming fibers before bundling, onemay first form the fibers by cleaving them from a larger material. InFIG. 8, a sheet of adsorbent material 70 is impregnated with a singleligand or receptor. That may be done by dissolving the compound in asolution and then impregnating a sheet of adsorbent paper (e.g. filterpaper). A crosslinking agent may be added to attach the receptor to thecellulose base of the paper or other support. Alternatively, one cancrosslink paper pulp to the receptor and then form the sheet of paper orfelt. That alternative technique provides a more consistent and uniformdistribution but requires greater amount of receptor. Either way, sheet(70) is produced. Many different sheets are prepared, wherein each sheetcontains a different receptor.

[0193] The sheets then are stacked together (like a book) with adhesiveand optionally an inert sheet (not impregnated, preferably black) as aspacer between each sheet of paper. That forms a book (71). One thentakes the book to a paper cutter or similar sectioning instrument and avery thin strip (72) is cut which resembles the “ribbon” of FIG. 1,object (2). The rest of the process is similar to that shown in FIG. 1.Multiple strips (72) from different books are stacked to form a bundle(73) that then is cut transversely to form a microarray (74). Anadhesive preferably is added to the ribbons to adhere them.Alternatively, an adhesive may be applied to a solid phase or the end ofthe bundle and the solid phase adhered to the bundle end beforesectioning.

[0194] Other films, which adsorb protein, such as nylon films, may beused. Inert films such as polyolefin, activated using heterobifunctionalphotoactivatable crosslinking reagents or simple polyurethane film suchas that of Thermedics may be used. One may use different proteins ondifferent sides of the sheet or film and separate the sheets with aninert sheet to separate cells (sectors) and signals in the finalmicroarray.

[0195] The fiber material is preferably glass, metal, plastic or otherpolymeric material. For coaxial composite fibers, the dissolvablecomponent may be made of a much wider variety of materials. Eachmaterial may be a composite of two or more components. The fibers mayact as light pipes or total internal reflection fiber optics to transmitpositional alignment and information regarding chemical and biologicalreactions occurring on the surface. The fiber material preferably ischosen to support attachment of cells and molecules of interest such asoligonucleotides, peptides and polysaccharides. Hollow fibers may beused to store cells in fresh, frozen or dried condition. Light andelectrons emitted directly or indirectly from a reaction or componentinside the fiber, particularly a hollow fiber such as a capillary, maybe amplified and easily detected when the fiber material is made ofglass or other transparent or translucent material. The fiber materialmay contain a component to react with, detect or convert into anotherform, the light, electrons or other chemical components emitting fromthe components or reactions occurring in the fiber. Detection ofchemiluminescent reactions in or on the fiber is a suitable method.

[0196] Gelling materials used in the present invention may be selectedfrom a large number of such known materials. Polymers such as agarose,gelatin, collagen, xanthene, carrageenan, alginate, or a thermosetting,thermoplastic, chemosetting or UV polymerizing polymer may be used.Non-polymeric gelling materials including waxes and clays may be used.Hydrogels are particularly preferred when a reaction occurring betweenthe agent of interest and an added substance for interrogation requiresan aqueous environment. The polymerizing agent or setting agent may beadded after the fiber has been cast by submerging the cast in a solutionof the agent or passing the agent along the outside of the fiber cast.

[0197] Hydrogels have many desirable features such as variable gelporosity, ability to bind proteins during or after polymerization, lownon-specific binding, transparency, harmless polymerization byproducts,controllable polymerization open time, usable with a variety of solventsand so on. Isocyanate polyurethane liquid prepolymers are preferred.

[0198] Those may be modified further by using thickeners, gums,hardening and crosslinking agents, plasticizers and various combinationsof gelling materials. In general, the gelling material should besufficiently inert to not interfere with an interaction between thebinding component and an analyte.

[0199] In the instant invention, an agent of interest is extracted intoan organic solvent, which is miscible with either a thermosettingplastic mixture, or one that is polymerized chemically or by UV orionizing radiation. That may be done by coating the agents withdetergents or other reagents, which will enhance solubility under theconditions chosen. The mixture then is extruded into long fibers or castinto fibers. The fibers would be identified by tags on the end of thefiber or by tags on the rolls carrying the fibers, and/or byincorporating different dyes therein. A barcode also may be printeddirectly near the end of fibers. Thermoplastic polymers may be used whenthe embedded product is sufficiently thermostable. Some of the fibersmay be colored differently to assist in the localization of specificligands in the array or to identify the array itself.

[0200] The solvent may be miscible in the gelling material or may beextractable or volatile to render a porous final product. Porousproducts are particularly preferred with solid filament fibers that areself-supporting.

[0201] The fibers or the gelling material thereof also may contain a dyeor other optical absorber so that only analyte/binding components on thesurface of each cell are visualized. Such an improvement reduces theeffects of diffusion rates through a gel or porous material that maychange with temperature, time, type of carrier liquid, etc. A dye thatadsorbs UV or emitted fluorescence will reduce fluorescence fromnon-surface analyte/binding component reactions.

[0202] Different dyes (fluorescent or non-fluorescent) may beincorporated into individual fibers. The permits the location of theindividual fibers in the two-dimensional array to be confirmed.

[0203] The solid filaments or capillary tubes comprising the fibers maybe adhered to each other by a variety of techniques. If the componentsare sufficiently heat stable, the fibers may be sintered together.Alternatively, a number of adhesives are known, including cyanoacrylateadhesives. The space between the fibers may be filled completely byadhesive or a monomer, which is polymerized. Thermoplastic and gellingmaterials also may constitute the adhesive by causing a large number offibers to be held together in a block. Even inert materials such asTeflon® tubes may have the surfaces thereof made reactive with sodiummetal in a hydrocarbon solvent to etch the surfaces. Non-chemical means,such as passing an electrical current through the fibers to fuse thefibers also may be used.

[0204] The open ends of the capillaries may be sealed against a flatplate, by pressing a deformable material against the surface,evaporating a plastic (e.g. paralene) on the surface, or by sealing witha chemical such as a thermoplastic or thermosetting plastic material.

[0205] There are two basic options for making two-dimensional arraysfrom such fibers. The first is to make and evaluate ribbons, and then toform a set of ribbons into a long rectangular bar, while the second isto make the bar at the outset, and then all of the fibers together inone step. The former option appears the more advantageous since theribbons can be evaluated individually before being formed into acomplete array. Once the two-dimensional array bar is formed, it can besectioned using conventional microtomes to form a very large number ofslices that can be attached, for example, to glass, metal, or plastic.Alternatively, one may first attach the solid phase material to the endof the bundle before sectioning the bundle. That may be performed byfirst coating either the end of the fiber bundle or the solid phasewith, if necessary, an adhesive such as a cyanoacrylate adhesive or apre-sectioning or post-sectioning sintering.

[0206] Dyed fibers would be visible in such arrays to confirmidentification and orientation. In addition, the fibers can be dyed insuch a manner that a visible pattern is formed if the array is madecorrectly, and the pattern may include a name or a number.

[0207] An advantage of the instant system is that very large numbers ofarrays may be cut, and some fraction used for standardization. Forexample, if a bar 100 cm in length were constructed, and if the bar werecut at 100-micron intervals, then 10,000 arrays would be available. Ifthe sections were 10 microns in thickness, then the number of arrayswould be 100,000.

[0208] If the individual fibers were 100 microns in diameter, therewould be 100 fibers per ribbon, and 10,000 in a bar of fibers with across-sectional area of 1 cm square. If there were 330 per ribbon, thenthe total number would be 108,900, approximately the number of expressedgenes postulated to be present in the human genome.

[0209] The instant invention is the first array to have such a largenumber of different cells per unit area on a microarray without thebinding agent being covalently attached to the chip. It is preferred forthe instant invention to have at least 100, more preferably 250, 500,1,000, 5,000, 10,000, 100,000 or a million or more cells per squarecentimeter of array. That is a much higher concentration thandepositable cells formed by microfluidics in commercial microarrays.

[0210] To increase greatly the number of cells per square centimeterbeyond even such high numbers, one may prepare a large fiber bundle withrelatively large fibers and stretch or draw the bundle. While that makesthe individual fibers thinner, the basic composition or orientation withrespect to each other and cross-section geometry will not be altered.That technique has the twin advantages of allowing one to make moremicroarrays and smaller microarrays. By using conventional 5 micronporous particles (as in the Example below) and a plastic embeddingmedium such as a low melting point wax, the result is deformable orductile fibers which may be drawn to very thin fibers of less than 20microns in diameter. The field of drawing thermoplastic materials iswell known per se. Even if not truly drawable through a die, one canpull or extrude plastic materials between rollers to lengthen and reducethe diameter of the fibers. With optional application of gentle heat,one need only pull the ends of the fiber bundle to generate the samelengthening and reducing of cross-sectional area. With smaller, porousparticles, the fibers may be drawn to even thinner dimensions therebypermitting microarrays of up to at least about 10 billion cells persquare centimeter of microarray.

[0211] In the field of fiber optics, bundles of optical fibers areheated and drawn into extremely thin optical fibers while retainingregistry within the bundle. Likewise, candy canes and candy withcross-sectional designs are prepared by drawing a large block. Evenglass beads used for hundreds of years also were prepared by suchtechniques.

[0212] High concentrations of cells (sectors) in a microarray have beenachieved using photolithography where the molecule of interest issynthesized on the microarray cell. However, the compounds generated byphotochemistry are limited. Further, chemically bound compounds interactdifferently from the same compounds when freely suspended. In abiological system, the active moieties may not be freely available forbinding. By contrast, the binding agents of the instant invention may bemerely entrapped in a matrix, fully retaining all chemical andbiological activity.

[0213] When using porous particles and immobilizing the molecule ofinterest inside the porous particle, it may be desirable to retain asuitable fluid inside the pores and use an immiscible embedding medium.In that arrangement, the embedding medium may be incompatible with themolecule of interest or use in a binding assay, yet still be useable.For example, an aqueous solution may be used to protect proteins and alow melting point wax used to embed the porous particles.

[0214] The known photochemical processes of Fodor et al., Nature364:555-6 (1993); Hacia et al., Molecular Psychiatry 3:483-92 (1998);and Fodor et al., Science 251:767-773 (1991) prepare short peptides andoligonucleotides covalently bound to the supporting chip. The process ofamino acid or nucleotide synthesis inherently limits the practicallength of the bound oligomer. Synthesis of entire proteins or genes onchips is not practical. Additionally, the secondary, tertiary andquaternary structure of the proteins may be important. By contrast, theinstant invention permits such.

[0215] Many different arrays ultimately may be required, and some,especially those developed for the identification of infectious agents,may need to be changed at frequent intervals. Further, newdisease-associated alleles will need to be incorporated into new arrays.To fill those requirements and allow changes and additions in arrays, itis important to have individual, stable fiber rolls available, and tohave the rolls unambiguously identified. Each roll may be identified bythe use of micro-stripes applied at short intervals along the roll. Inaddition, different tubes may have different colors, and non-fluorescentdyes incorporated into the gels to serve as identifiers, or bar coding,may be printed on individual fibers.

[0216] Not only can the chips of the instant invention be used toidentify infectious agents by identifying characteristic nucleic acidsequences, for example, the chips also can be used for identifyingintact bacteria, mycoplasmas, yeast, nanobacteria and viruses usingarrays of immobilized specific antibodies.

[0217] The invention may be used for the identification of viruses orother infectious particles isolated by microbanding tubes. Suchmicrobanding tubes are particular centrifuge tubes of stepped decreasingdiameter from the open end to the closed end of the tube that enableconcentration of desired low concentration biological elements in asmall volume following appropriate methods of centrifugation. See, forexample, WO099/46047. Thus, microbes from biological samples, e.g. serumor plasma, may be concentrated, stained with a fluorescent nucleic acidstain such as TOTO-1 or YOPRO-1, and then allowed to find matchingantibodies on the array. They then may be detected by scanning forfluorescence and identified by position. It is equally a part of theinstant invention to immobilize microorganisms or other molecules ofinterest in the described chips, to use such chips to localizeantibodies from body fluids, and then to discover the location of thelatter using a fluorescent anti-human antibody, thus diagnosing thedisease which elicited antibody production.

[0218] Because the bundle is maintained, additional fibers or ribbonsmay be added to the bundle as needed before sectioning additionalarrays. That allows one to detect and measure newly discovered emergingdiseases, new proteins, genes or compounds without recreating acompletely new bundle.

[0219] The invention may be applied in an alternative fashion in whichthe bundles are stored at user sites, and the arrays sliced as needed.That arrangement may be useful for research purposes where identicalarrays are required over the long term, but only a few are required atany one time.

[0220] Another alternative to slicing the bundle and using the sectionsthereof as separate microarrays is to perform the assay with the end ofthe bundle directly. After the assay is performed wherein a first samplecould be applied to the cut cross-sectional surface, and washed off, adetector could image the result. One then may mount the bundle in amicrotome device, if the assay were not already so mounted before theassay. A blade then could remove the used surface of the bundle,exposing a fresh surface for the next assay, which would repeat the samesteps. The bundle thus could be used in one machine for a series of upto 100,000 or more assays performed one after another. That arrangementhas certain advantages as optical or electrical detection may beperformed through the bundle itself with fiber optic fibers orconductive fibers. The detection system may be attached continuously tothe bundle while a more general light or electrical energy applied tothe end being used for testing. Specifically note FIG. 6 where thetesting technology may be adapted to a detection system.

[0221] The invention also allows different immobilization technologies,different classes of immobilized agents of interest, different classesof analytes and different types of detection methodologies to beemployed on the same chip.

[0222] Since channels are reproducible between plates, the location ofeach channel or cell may be determined accurately by mechanical means.Reference markings on polished edges or other suitable locations furtheridentify each cell in the array. Current commercially available computerdriven two-dimensional drives of sufficient accuracy can be used so thateach cell may be visualized or tested individually, or material may beadded thereto or withdrawn therefrom.

[0223] Cut surfaces of each plate may be polished so that matchingplates may be opposed to each other with little possibility of crossleakage. Surface treatment with a material repellant to the fluid to beeventually located inside each cell further reduces cross leakage. Forexample, fluorinating (Teflonizing) or silanizing agents repel waterthereby generating sufficient surface tension to reduce cross leakage ofcells filled with an aqueous solution.

[0224] After sections have been cut from a bundle, the sectionsgenerally are bound to a solid backing to provide structural support andease of handling. The solid backing is typically a sheet of plastic ormetal although other materials may be used. The attachment generally isdone by a permanent adhesive or heat fision.

[0225] Individual cells in the array may be detected or visualized byscanning the entire array or portions thereof (e.g. one or a few cells)with a charged coupled device (CCD) or by illuminating one or a fewchannels at a time, such as by a condenser lens and objective lens. Theabsorbance and emission of light thus may be detected. An optical fiberbundle aligned and registering with the microarray may be used foroptically detecting differences between the cells of the microarray.

[0226] Detection may be based on a large number of detectable labelsincluding radioactive, enzyme, luminescent, electroluminescence,optically absorbent dye, magnetic, spin-labeled, oxidizers or reducers,chemiluminescence, or indirect labels which interact with a detectablecomponent interacting with the agents of interest in the microarray.Detection may also be accomplished by measuring surface plasmonresonance, see U.S. Pat. No. 5,955,729. A suitable detectable labelingsystem is based on fluorescence, usually epifluorescence. That requiresthat the interrogating sample be labeled with one or more fluorescentdyes. The amount of test material required is very small since the dyewould be applied to the arrays as a thin dilute film. Hybridization ofnucleic acids would be done under conditions of carefully controlledstringency.

[0227] To distinguish selected channels, one either may seal off theselected channels and/or fill the channels with an easily detectablesubstance. Different colored inks, dyes and colored materials areparticularly well suited as well as detectable components similar to oropposite from the detectable component(s) being detected in other cells.Printing methods with drying inks or plastics, sublimation, solventcontaining an ink, or ink-jet printing may be used. The indicia soformed permits better alignment or easily detectable marking when thearray is in use. That permits easy optical alignment.

[0228] Once the microarray has been used in a binding assay and theligainds are bound to the receptors, in certain instances it may beuseful to provide further identification of the ligand. In certainsituations, one does not know the entire structure of the ligand fromthe receptor that specifically binds to it. For example, if the ligandis a cell, a macromolecular complex or a derivatized molecule with thederivatized portion acting as the ligand, etc., further analysis may bedesirable. In that situation, one may elute the ligands from themicroarray and collect the ligand for further analysis. Forantibody/antigen binding, a pH 2-3 environment or other conditionsshould strip the ligands. For nucleic acid hybridization, raising thetemperature should strip the ligands. A variety of other chemical,physical and electrical techniques for breaking such bonds are known perse.

[0229] To enhance specificity to the elution process, the substrate canbe configured to enable maintaining a charge that would enhance trappingthe biological agent of interest at a particular cell (sector). Forexample, if the agent of interest is a nucleic acid, each cell can beconfigured to carry a positive charge. A counterelectrode carries theopposite charge. Then, if necessary, a particular medium is placed intothe cell and the charges in the electrodes reversed thereby releasingthe ligands, in the example, nucleic acids, at that location. Thecounterelectrode also may be part of or contain appended thereto amicropipette to collect the elements released from the cell, see U.S.Pat. No. 5,434,049. Preferably, one uses a porous membrane and applies acurrent on opposite sides of the membrane.

[0230] The method used for analysis of the eluate may be capillaryelectrophoresis, mass spectrometry or a second binding assay. Convenientto mass spectrometry, the microarray itself may be introduced into alaser-matrix desorption system incorporated into a mass spectrometrysystem wherein bound molecules are desorbed and analyzed.

[0231] Once the analytes have been striped from the microarray, themicroarray may be reused. That reuse process has the advantage of beingstandardized by multiple controls over time.

[0232] Additionally, if the receptor is attached to the matrix of themicroarray by a cleavable linker, one can isolate the analyte bycleaving the linker. Different cells of the microarray may havedifferent linkers or the same linker and subsequent purification may beneeded before additional analysis.

[0233] The previous methodology for preparation of protein chipsrequires preparation, use and reuse of large numbers of proteins insolution. Proteins, nucleic acids, biological cells, other chemicals andcomplexes in solution are unstable and deteriorate over time. Even iffrozen, repeated use may involve repeated freeze-thaw cycles thatdenature certain proteins as well. By contrast, immobilized proteinshave been shown to be stable over long periods of time.

[0234] For the purposes of the instant invention, the term “substrate”refers to the glass capillary arrays with “major surfaces” referring tothe open ends of the channel plate and “binding reagent” refers to theDNA, protein or antibody (collectively macromolecules),cells/microorganisms/cellular systems or other agent of interest.

[0235] The following examples are included for purposes of illustratingcertain aspects of the invention and should not be construed aslimiting.

EXAMPLE 1 Formation and Analysis of a Microarray

[0236] Antibodies were prepared by affinity purification by reversiblebinding to the respective immobilized antigens and subsequentlyimmobilized on particulate supports (Poros G, made by PE Biosystems) inan Integral 100Q biochromatography workstation.

[0237] Each antibody support was made by trapping the antibody on acolumn of Poros G (commercially available Poros particles pre-coatedwith protein G, a bacterial protein capable of binding manyimmunoglobulins by the Fc domain) and subsequently cross-linking theantibody and the protein G with dimethylpimelimidate (following the PEBiosystems protocol) to immobilize the antibody covalently on the Porosparticles. Such antibody columns can be reused (with an acid elution ofbound antigen) more than 100 times in a subtractive mode, and thereforeare extremely stable. Each antibody support was characterized todemonstrate specificity for a single antigen.

[0238] Antibodies directed against human serum albumin (HSA),transferrin (Tf), and haptoglobin (Hp) were used. A mixture of the threesupports was made for use in serum subtraction. A total of threesupports were used in tests with: 1) rabbit anti HSA, 2) rabbitanti-human Tf and rabbit anti-human Hp and 3) mixed anti-HSA, Tf and Hp.Unmodified BA Poros (commercially available streptavidin coated Poros),was used as a non-antibody control. Thus, a total of four supports wereused.

[0239] Poros particles are roughly spherical and highly reticulated(with many internal crevices), having a diameter of approximately 5microns. Attached proteins are distributed over the internal surfaces aswell as the exterior surface of the particle. By embedding the particlesin a suitable medium, a sliceable solid matrix in which the antibody wasimmobilized and fairly uniformly distributed was created. By exploitingthe 3-dimensional nature of the support, a slice containing suchparticles offers greater capacity (for antibody and thus for antigenbinding) than a simple flat surface as used in current microarrays.

[0240] Each of the four types of antibody-bearing particles was mixedwith an approximately equal volume of 0.75% agarose melted inphosphate-buffered saline (PBS). The agarose for the rabbit anti-HSAbeads contained green food coloring. Likewise, the anti-Tf and Hpagarose were colored blue, the mixed anti-HSA, Tf and Hp agarose wascolored yellow and the Poros BA containing agarose was white(uncolored). Each melted agarose/bead combination was sucked into alength of one mm diameter plastic tubing of 10 cm in length attached toa 1 ml syringe and plunged in ice water. In several minutes, the agarosegelled into a jelly-like rod containing approximately 50% Poros beads byvolume. The four rods thus obtained (each containing one of the fourbead types above with a different protein coating) were laid into analuminum channel with more melted agarose to form an array of 2×2parallel rods embedded in a square cross-section bar of agarose.

[0241] After the bar gelled, the gel was removed from the aluminumchannel mold, and transverse sections were prepared by slicing thinslices perpendicular to the axis of the bar (and the filaments) andmounted on a glass slide. The sections revealed a pattern of 4 circularareas (the filaments) containing embedded particulate material (carryingimmobilized protein) surrounded by clear embedding matrix of agarose bymicroscopy. The circular zones of embedded beads were more stable anddid not split.

[0242] To test specific protein binding to the beads in the fourcircular zones of a section forming the microarray, commerciallyavailable HSA and Tf protein were labeled with fluoresceinisothiocyanate (FITC) on Cellite (from Sigma). Cellite is a commercialcarrier for insoluble FITC. The proteins were dissolved in about 4 ml of0.4M sodium bicarbonate buffer (˜pH 8.3) and added to the dry FITC onCellite in the following amounts: ˜4.5 mg HSA 30 mg FITC on Cellite ˜2.8mg Tf 18 mg FITC on Cellite ˜4.5 mg Serum Protein (20 μl) 10 mg FITC onCellite

[0243] The reaction was conducted at room temperature for 30 minutes.The Cellite was removed by centrifugation, and the supernatant proteinand unreacted dye placed in a centrifugal protein concentrator, wherethe protein was washed by repeated dilution and re-concentration inbuffer. The fluid was centrifuged to remove the Cellite and supernatantrecentrifuged with 4 ml sodium bicarbonate buffer until clear.

[0244] Sections of the 4-filament array were laid flat on a glassmicroscope slide and exposed to a solution of fluorescently labeled HSA.During the exposure of the section, the protein was expected to interactspecifically with the antibodies present on two filaments (round areason the section): the two filaments were those bearing antibodies to HSAand the mixed anti-HSA, Tf and Hp. Labeled HSA was not expected tointeract with the filaments carrying antibodies to Tf alone or to thefilament carrying streptavidin alone.

[0245] The sections were examined under an epifluorescence microscopeequipped with a 500 nm low pass filter and a 510 nm high pass filter forfluorescein fluorescence detection and a 35 mm camera.

[0246] Prior to extensive washing, all four circular Poros zones showedbright fluorescence, with no discernable differences. The fact that thePoros zones showed higher fluorescence than the agarose matrixsurrounding the filaments is an indication that the pores of the Porosparticles remained unclogged and that the particle-containing zones thusallowed freer diffusion of labeled HSA into the sections.

[0247] The sections then were washed extensively in PBS and reexaminedunder the fluorescence microscope. The resulting images, captured on 35mm color slides, demonstrate that after washing, the labeled albuminspecifically bound to the two filaments containing HAS antibody and wasremoved from the other two, thus establishing the ability of thesections specifically to detect an individual protein. The twospecifically labeled filaments were diagonally opposite one another inthe 2×2 array, which was consistent with the diagonally oppositepositions of the anti-HSA and mixed anti-HSA, Tf and Hp agarosefilaments.

EXAMPLE 2 Manufacture and Use of Diagnostic Array DetectingAutoantibodies to Mitochondrial or Lysosomal Proteins

[0248] Suspensions of whole isolated rat and mouse liver mitochondria,lysosomes and expressed proteins are suspended or dissolved in anaqueous buffer, at 10 mg/ml concentration, and optionally fixed withglutaraldehyde (1%). One ml of each preparation is mixed according tothe kit instructions with 20 ml of JB-4 (Polysciences) catalyzedinfiltration resin prepared by mixing 20 ml of monomer A containing 0.17g of catalyst. After complete mixing, 40 ml of monomer B containing 0.17g catalyst is added with stirring. When completely dissolved, 0.8 g ofAccelerator is added, the mixture placed in a syringe and injected into0.0625 inch internal diameter Teflon tubing under anaerobic conditions.Polymerization occurs at room temperature in approximately 50 minutes.The ends of the tubes then are heat sealed and stored cold until used,or are immediately extruded for use in preparing a fiber bundle. Bundlesare prepared by laying 10 or more fibers in parallel, to make asingle-layered array, in an elongated Teflon box. Additional JB-4 resinwithout protein then is poured in, the box briefly evacuated to removeair bubbles and the resin allowed to set. Several such flat arrays thenmay be stacked in parallel to make a three-dimensional grouping, and thewhole grouping further vacuum impregnated to form a three-dimensionalbundle. After polymerization, the bundle is cut with a steel or glassmicrotome knife to give sections 5-20 microns thick and the sectionsplaced on glass slide. The sections are mounted using Plastic Mount®, orare dried and mounted with Poly-Mount® (Available from Polysciences).

[0249] Tests for autoantibodies are done by placing 0.25 ml of a 1:10dilution of human serum on each chip and incubating the arrays at 25° C.for 20 minutes. The arrays then are rinsed in phosphate buffered salinefour times, and then are immersed in a solution of goat anti-humanglobulin conjugated with horseradish peroxidase. After a further 20minute incubation, the arrays again are washed four times with buffer,and then placed in a solution of 3,3′,5,5′-tetramethylbenzidine in anorganic base to which is added a hydrogen peroxide solution (0.02%) in acitric acid buffer. An insoluble blue color indicates the presence ofautoantibodies.

EXAMPLE 3 Manufacture and Use of a Diagnostic Array Using HistologicalEmbedding Support

[0250] Arrays are prepared which incorporate fixed infectious particlesto be used to detect convalescent antibodies appearing late in thehistory of an infection. That is important in following sentinelpopulations to determine what infections are occurring.

[0251] Immuno-Bed GMA water-miscible embedding medium is made up asdirected (Polysciences Inc.), and small batches are mixed with differentsuspensions of fixed selected viruses (average titer 10⁹/ml) or fixedbacterial cells (average 10⁷ particles/ml). The suspension is placed ina syringe and forced under pressure into Teflon® tubing of {fraction(1/16)}-inch internal diameter and allowed to polymerize at roomtemperature. The tubing is pre-treated with metallic sodium in anorganic medium to provide a surface, which will adhere to epoxy resins.The polymerized fiber is stored in the coiled Teflon® tubing in thecold.

[0252] The arrays are assembled in bundles using jigs to hold the fibersin parallel array, after which the array is infiltrated with an epoxyresin. The finished bundle, which includes sections of Teflon® tubing,is sectioned and the sections mounted on glass slides using an epoxyresin mounting medium. The sections are washed for rehydration and thenare exposed to convalescent antisera. The chips then are extensivelywashed and exposed to goat anti-human IgG with the covalently attachedfluorescent dye fluorescein. Identification of convalescent antibodiesis done by detecting and measuring fluorescence using a CCD camera.

EXAMPLE 4 Manufacture of Diagnostic Array Using Sintered Strips

[0253] Sintered polystyrene sheets {fraction (1/16)} inch thick are cutinto square cross-section strips and each exposed to dilute solutions ofone monoclonal antibody to a series of infectious agents includingviruses such as rhinoviruses, herpes simplex viruses, influenza virustype A, respiratory syncytial virus, varicella-zoster virus(chickenpox), mycobacterium tuberculosis, cytomegalovirus, Epstein-Barrvirus, Hepatitis B Virus (surface antigen and separately core antigen)poliovirus (three strains) and others. The strips are rinsed, dried andglued together with an acrylonitrile adhesive to form athree-dimensional array that is sectioned to produce arrays 5-100microns thick. Biological samples containing infectious viruses fromindividuals with viral diseases are fluorescently stained with thenucleic-acid specific dye YOYO-1 (Molecular Probes) and isolated andconcentrated using centrifugal microbanding, see WO99/46047 supra, toconcentrate the infectious particles into microliter volumes. Theconcentrated viruses are applied to the array and are agitatedmechanically to move the virus particles over the array for one hour.The array then is washed, excess fluid removed by suction andilluminated with ultraviolet light at 490 nm. The image is captured withan Apogee CCD camera using a 520 nm filter. Quantitative data isobtained from the processed image using the PMIS image analysis program.

EXAMPLE 5 Manufacture and Use of Diagnostic Array Having ImmobilizedOligonucleotides

[0254] Polystyrene beads (10-50 microns in diameter) from solid phaseoligonucleotide synthesis with oligonucleotides covalently attached aresuspended in buffer and packed into hollow glass fibers of 500 micronsinternal diameter under hydrostatic pressure initially and then underair pressure up to 500 psi to expel the supporting liquid. The fiberthen is heated briefly under controlled conditions to partially sinterthe contents. An array of fibers then is prepared following the methodsin the examples above, embedded in a low viscosity epoxy resin withintermittent vacuum to remove air bubbles and then allowed to set. Thebundle is sectioned using a diamond saw. The array is used in aflowthrough arrangement so that the materials thereon can be manipulatedin a fashion similar to that conducted with larger multiwell microtiterplates as described in U.S. Pat. No. 5,843,767, supra

EXAMPLE 6 Manufacture of Multiwelled Plates

[0255] Commercially available glass capillary arrays (GCA)(Galileo) arein the shape of a thin disk having 2.5 cm×2.5 cm×0.5 mm thickdimensions. The GCA has approximately 50% of the area composed of 50μholes or approximately 156,000 holes having a total volume ofapproximately 0.1 ml. The bottom surface of the GCA is glued to aTeflon® sheet with cyanoacrylate adhesive (SUPERGLUE).

EXAMPLE 7 Cloning and Replica Plating in Glass Capillary Arrays

[0256] A colony of Streptococcus pyrogenes Group A and a colony of GroupB were picked from a plate and mixed together in nutrient agar forming asuspension of the bacterial cells (other microorganisms, animal or plantcells are equally applicable) and are diluted to an approximateconcentration of 20,000 cells/ml of culture medium. About 0.1 ml of thesuspension is applied to the surface of the GCA. That yields about 1cell per 100 holes to ensure only single cell clones result. The GCA isplaced in a sterile petri dish, covered and incubated overnight at 37°C.

[0257] Two additional sterile GCA's without a Teflon® sheet on thebottom are filled with 0.1 ml heated liquid culture fluid supplementedwith 1% agarose, cooled until almost solidified and stacked directly ontop of the GCA having cloned bacterial cells so that the holes from eachGCA are in register. A top sheet of Teflon® is pressed on tightly andthe stack is clamped together. The entire stack is turned upside downand incubated for five minutes at room temperature. The entire stack isturned sideways and incubated overnight at 37° C. The stack then isturned upright, unclasped and individual GCA's are separated. Theoriginal GCA is retained for fiiert use.

[0258] Each of the two added GCA's is placed in a glass flask, attachedto a lyophilizer and vacuum dried for 1 hour. The GCA's are removed and0.1 ml of FITC conjugated antibody to Streptococcus Group A (DIFCO) isadded to each GCA and incubated at room temperature for 10 minutes. EachGCA then is blotted on an adsorbent tissue (KIMWIPE) to remove fluid.The microarray is washed by submersion in PBS and blotted dry again. Thefluorescent holes in the GCA's and bacteria containing holes in theoriginal GCA are detected using a CCD scanner which gives 12.5μ pixelsand is capable of a resolution of 25μ needed to detect holes whichcontain cell clones.

[0259] The scanner is first set to scan for fluorescence and then forabsorbance to detect the presence of bacterial clones. Absorbance isused to indicate presence of bacteria to align the holes of the twoGCA's. Fluorescence is detected in some but not all of the holescontaining bacterial clones in the original GCA and correspond topresence of Group S bacteria.

EXAMPLE 8 Selecting Monoclonal Antibodies

[0260] Monoclonal antibody-secreting hybridomas in suspension arediluted to approximately 20,000 cells/ml RPMI 1640+5% fetal bovine serumculture solution and 0.1 ml is added to the GCA of EXAMPLE 6 and themethod of EXAMPLE 7 repeated except for incubation being at 37° C. in aCO₂ incubator for two days and the GCA's being pretreated with 10% fetalcalf serum for 30 minutes. An additional GCA is filled with protein-freesaline solution, stacked and clamped. The stack is not turned at all butincubated at room temperature for 15 minutes, unclasped and then vacuumdried as before. About 0.1 ml of FITC-conjugated goat anti-mouseimmunoglobulin is added to the additional GCA, incubated, removed,washed and scanned for fluorescence as before. Antibody secretinghybridomas are deduced from the location of fluorescence on the GCA.

EXAMPLE 9 Screening Libraries of Proteins for Biological Properties

[0261] Human serum proteins are separated by 2-dimensionalelectrophoresis as per Baekkeskov et al., Diabetes 38(9): 1133-41(1989). Two hundred spots are punched from the gel and the individualproteins dialyzed in 1 ml of PBS. One ml of the protein solutions ismixed with 40 mg of acrylamide monomer with catalyst and pumped into 1mm internal diameter, one meter long polypropylene tubes, the ends heatsealed and each tube tagged. A number of control tubes are prepared withvarious dyes for easy identification of the correct orientation of themicroarray when formed. The acrylamide is allowed to polymerizeovernight. The tubes are aligned in a bracket and glued between rows asabove. The bundle is cut by a microtome under freezing conditions into10 micrometer thick slices and the microarray is immediately fixed on aplastic sheet.

[0262] Mouse monoclonal antibodies to the following antigens (VectorLabs) are individually contacted to a separate microarray, incubated,washed, dried and followed by contacting with FITC-conjugated(fluorescein-labeled) goat anti-mouse IgG and scanned as in EXAMPLE 8above. Insulin, calcitonin, glucagon, epidermal growth factor,interferon, CEA, prostatic acid phosphatase and human IgG are among thecommon antigens tested. Both hormone levels and tumor antigen levels aredetermined in a semi-quantitative manner.

EXAMPLE 10 Rapid Antibiotic Sensitivity Testing

[0263] Microarrays are prepared in accordance with EXAMPLE 2 except forfilling each tube with nutrient agar mixed with various antibiotics inthe following configuration. Five two-fold dilutions across theeffective spectrum of useful concentrations of the antibiotics,erythromycin, penicillin V, tetracycline, ampicillin,trimethoprim/sulfamethiozole, cefaclor, ofloxacin and nitrofurantoninand 10 two-fold dilutions of 34 new compounds, each a candidate for useas an antibiotic are used.

[0264] A colony of an unknown sample of E. coli grown from urine of apatient was suspended in 1 ml nutrient broth supplemented with eitherfluorescein acetate or trypan blue and placed on each of two microarraysand incubated at 37° C. The microarray is scanned for fluorescence andfor absorbance at the beginning and after 30 minutes incubation.Microarray cells with detectable increases in fluorescence (scannedfluorescence minus fluorescence from initial scanning) were consideredto have growing cells. Microarray cells with increases in trypan blueabsorbance from the beginning to 30 minutes were considered to have deadcells. Minimal inhibitory concentrations (MIC's) and minimalbactericidal concentrations (MBC's) thus were determined. The possibleeffectiveness of the new candidate compounds likewise was deduced.

[0265] Another 1 ml of saline containing another suspended colony of theunknown sample of E. coli was plated on conventional Mueller-Hintonplates with antibiotic disks and incubated overnight. MIC's weredetermined the next day based on the diameter of the zone of inhibition.The MIC's from the microarray are comparable to standardized growthinhibition measurements. For example, for nitrofurantonin, the zonediameter from a 300 mcg disk in millimeters is >17 mm susceptible, 15-16mm intermediate and <14 mm resistant which corresponds to a MIC inmcg/ml of <32, 64 and >128 respectively. Two-fold dilutions ofnitroflrantonin in the microarray are at 16, 32, 64, 128 and 256 mcg/ml.

[0266] The method is repeated with known strains of E. coli having knowndiffering levels of antibiotic resistance and with many different commonmicroorganisms with different levels of antibiotic resistance. Theresults indicate which of the 34 candidate compounds are to be testedfurer as potential antibiotics.

EXAMPLE 11 Anticancer Diagnostic and Drug Screening

[0267] Microarrays are prepared according to the method in EXAMPLE 2with suspensions of various fresh cells from a leukemia patient, severalleukemia cell lines (HTB, ATCC), normal peripheral white blood cells andnormal bone marrow cells. The microarrays are treated by analkaline-lysing and protease K-digesting reagent, heat denatured and adigoxigenin-labeled DNA probe for the following genes: N-myc, C-myc,K-ras, p53, HER-2/neu and a candidate DNA probe for diagnostic purposes,are applied thereto. Texas Red-labeled anti-digoxigenin antibody isadded and the pattern and amount of binding are determined.

EXAMPLE 12 Hepatitis Testing

[0268] It is desirable to know the type of viral hepatitis and the stageof infection to best treat a patient. A microarray is prepared as inEXAMPLE 2 except that ten, 2-fold dilutions of mouse monoclonalantibodies to HAV, HBsAg, HBcAg, HCV, HDV and HEV and 2-fold dilutionsof the same antigens are used. Three tubes of each are prepared and usedin the microarray along with a pattern of controls. Approximately threedrops of serum sample is contacted with the microarray, incubated in a37° C. water bath for 10 minutes and washed four times with PBS. About 1ml of a reagent of fluorescein-labeled monoclonal antibodies tonon-overlapping epitopes of each of the antigens, fluorescein-labeledmouse anti-human IgG and rhodamine-labeled mouse anti-human IgM is addedto the microarray, incubated for 10 minutes in a 37° C. water bath andwashed four times with PBS. The microarray is scanned for fluorescenceat both the wavelength of fluorescein and rhodamine emissions and theresults determined for which cells of the microarray demonstratefluorescence, the wavelength of light and the level thereof.

[0269] The microarray is designed for both initial diagnosis and formonitoring treatment and remission by detecting antigens and antibodiesin convalescent serum. Two-fold dilutions and measuring the level offluorescence at each cell provide quantitative results.

EXAMPLE 13 Screening Active Compound Candidates

[0270] Microarrays are prepared according to EXAMPLE 2 except 380 newcandidate compounds are introduced into the fibers. Three drops of asolution containing the glutamate receptor 2 are added to the microarrayfollowed by incubation at 37° C. for 10 minutes. The microarray iswashed and dried as before. A 1:10 dilution of mouse monoclonal antibodyto glutamate receptor 2 (Vector Labs) is added, incubated, washed anddried as before. FITC-conjugated goat anti-mouse IgG is added and themicroarray scanned.

[0271] Fluorescent cells correspond to compounds that bind to thereceptor. Since the receptor is involved in learning, memory, seizuresand other neurological conditions, by binding the neurotransmitterglutamate, both agonists and antagonists are of pharmacologicalinterest.

EXAMPLE 14 Formation and Analysis of a Microarray by Fluorescence

[0272] A microarray was prepared from cylindrical polymethacrylatefibers containing a) microbeads with immobilized antibodies to rat IgG,b) microbeads with immobilized antibodies to human IgG and c) nomicrobeads as a control. The array was formed by aligning the fibers inparallel along the long axis, sectioning with a microtome, thentransferring the sections to glass slides. The slides there were testedin a fluorescent immunoassay to demonstrate specific protein binding tothe beads as follows:

[0273] Two disposable columns, each containing about 0.5 ml of UltraLinkImmobilized Streptavidin Plus beads (50-80 microns diameter, with acapacity of 10 mg of biotin-BSA per ml of beads, Pierce Chemical Co.,Rockford, Ill.), were washed with phosphate buffered saline pH 7.2containing 0.05% sodium azide. The slides were treated sequentially withfive 1 ml solutions containing 0.5 mg of biotin labeled goat anti-humanIgG on one column and 0.5 mg of biotin labeled goat anti-rat IgG on theother column. The columns there were treated with excess biotin,followed by washing with PBS.

[0274] The embedding material used was ImmunoBed (Polysciences, Inc.,Warrington, Pa.) prepared according to the directions of themanufacturer. Dry catalyst (225 mg) was dissolved in 25 ml of ImmunoBedSolution A. To that solution was added 1 ml of hmmunoBed Solution B. Themixture was kept cold and then introduced into a four foot length ofTeflon tubing ({fraction (1/32)} inch ID) using a syringe attached tothe tubing. The tubing filled with ImmunoBed resin was allowed to standundisturbed overnight at room temperature. The polymerized fiber couldbe removed from the Teflon tubing by trimming the end of the tubing witha single edge razor blade to expose the fiber, then gently pulling thefiber from the tubing.

[0275] UltraLink beads containing antibodies to human IgG and rat IgGwere prepared as described above. About 0.5 ml of each were collected bycentrifugation at 2000 rpm for 10 minutes then mixed with 5 ml of coldImmunoBed solution (Solution A+catalyst+Solution B) prepared asdescribed above. The beads then were centrifuged for 10 minutes at 2000rpm at 5° C. That was repeated three times. The pelleted beads then wereresuspended in 1 ml of the ImmunoBed solution and drawn into {fraction(1/32)} inch ID Teflon tubing. The tubing was folded into a bundle,placed in a centrifuge bucket and then centrifuged for 10 minutes at2500 rpm. The buckets were removed and left overnight at roomtemperature to allow the ImmunoBed to polymerize. The bundles were cutinto sections by cutting the top end of the folds and the strands wereextruded.

[0276] Two control fibers and two experimental fibers were cut tolengths of 1.5 cm each. The fibers were aligned along the long axis andplaced in a groove in a Teflon block. A glass slide was placed over thefibers and clamped in place such that about 1 mm of each of the fiberswas exposed. ImmunoBed solution (Solution A+catalyst+Solution B) wasintroduced to the exposed tips of the fibers and allowed to flow underthe glass slide to fill the space around and between the fibers. Thestructures were left overnight at room temperature to allow completepolymerization. The array was removed from the mold and sectioned in aLeica Model RM-2155 Microtome. Thin sections (10 microns) weretransferred to a glass slide containing a 20 μl droplet of water and thewater was allowed to evaporate at room temperature. That left thesections firmly attached to the glass slide. Sections of 50 micronsthick give more background fluorescence.

[0277] The 10-micron section prepared above and mounted on a glass slidewas treated with 100 μl of normal rat serum (IgG containing), diluted1:50 with PBS containing 1 mg/ml BSA, for 60 minutes at roomtemperature. The solution was drained from the slide, rinsed 1 time with100 μl PBS/BSA, then washed three times with 100 μl PBS/BSA for 5minutes before draining. After the last wash, 100 μl ofR-Phycoerythrin-labeled affinity purified goat antibody to rat IgG (H+L)(Kirkegaard and Perry, Gaithersburg, Md.), diluted 1:100 with PBS/BSAwere added and allowed to stand for 60 minutes at room temperature. Thesolution then was drained and washed 4 times as before. Afterfluorescent immunostaining, the section was viewed in an Olympus ModelBX-40 fluorescent microscope (Olympus America, Inc., Melville, N.Y.)using a green filter (exciter filter 510-550 nm, barrier filter 590 nm).The four circular slices that comprised the 10-micron slice included 2control slices, one slice containing beads with anti-human IgG and oneslice containing beads with anti-rat IgG. The circular slice containingantibody to rat IgG was more highly fluorescent than the slice thatcontained anti-human IgG, and the 2 control slices, thus demonstratingthe specificity of the reaction. Table of Data Microarray Fiber Amountof Fluorescence Content Observer #1* Observer #2** Antibody to Rat IgG++++ 10  Control (no antibody) 0 0 Antibody to Human IgG ++ 3 Control(no antibody) ++ 2

EXAMPLE 15 Detection Method for Microarray Using Rare Earth or HeavyMetal Conjugate Dyes

[0278] Antibodies are prepared by affinity purification by reversiblebinding to the respective immobilized antigens and subsequentlyimmobilized on particulate supports (Poros G, made by PE Biosystems) inan Integral 100Q biochromatography workstation.

[0279] Each antibody support is made by trapping the antibody on acolumn of Poros G (commercially available Poros particles pre-coatedwith protein G, a bacterial protein capable of binding manyimmunoglobulins by the Fc domain) and subsequently cross-linking theantibody and the protein G with dimethylpimelimidate (following the PEBiosystems protocol) to immobilize the antibody covalently on the Porosparticles. Such antibody columns can be reused (with an acid elution ofbound antigen) more than 100 times in a subtractive mode, and thereforeare extremely stable. Each antibody support is characterized todemonstrate specificity for a single antigen.

[0280] Antibodies directed against human serum albumin (HSA),transferrin (Tf) and haptoglobin (Hp) are used. A mixture of the threesupports is made for use in serum subtraction. A total of three supportsare used in tests with: 1) rabbit anti HSA, 2) rabbit anti-human Tf andrabbit anti-human Hp and 3) mixed anti-HSA Tf and Hp. Unmodified BAPoros (commercially available streptavidin coated Poros), is used as anon-antibody control. Thus, a total of four supports were used.

[0281] Poros particles are roughly spherical and highly reticulated(with many internal crevices), having a diameter of approximately 5microns. Attached proteins are distributed over the internal surfaces aswell as the exterior surface of the particle. By embedding the particlesin a suitable medium, a sliceable solid matrix in which the antibody isimmobilized and fairly uniformly distributed is created. By exploitingthe 3-dimensional nature of the support, a slice containing suchparticles offers greater capacity (for antibody and thus for antigenbinding) than a simple flat surface as used in current microarrays.

[0282] Each of the four types of antibody-bearing particles is mixedwith an approximately equal volume of 0.75% agarose melted inphosphate-buffered saline (PBS). The agarose for the rabbit anti-HSAbeads contained green food coloring. Likewise, the anti-Tf and Hpagarose are colored blue, the mixed anti-HSA, Tf and Hp agarose iscolored yellow and the Poros BA containing agarose-is white (uncolored).Each melted agarose/bead combination is introduced into a length of onemm diameter plastic tubing of 10 cm in length attached to a 1 ml syringeand plunged in ice water. In several minutes, the agarose gels into- ajelly-like rod containing approximately 50% Poros beads by volume. Thefour rods thus obtained (each containing one of the four bead typesabove with a different protein coating) are laid into an aluminumchannel with melted agarose to form an array of 2×2 parallel rodsembedded in a square cross-section bar of agarose.

[0283] After the bar gels, the gel is removed from the aluminum channelmold and transverse sections are prepared by cutting thin slicesperpendicular to the axis of the bar (and the filaments) and the slicesare mounted on a glass slide. The sections revealed by microscopy apattern of 4 circular areas (the filaments) containing embeddedparticulate material (carrying immobilized protein) surrounded by clearembedding matrix of agarose. The circular zones of embedded beads arestable.

[0284] To test specific protein binding to the beads in the fourcircular zones of a section forming the microarray, commerciallyavailable HSA and Tf protein are labeled with either europium chelate orruthenium chelate using either SYPRO RUBY or SYPRO ROSE protein stain asdirected by the manufacturer (Molecular Probes Inc., Eugene, Oreg.).

[0285] Sections of the 4-filament array are laid flat on a glassmicroscope slide and exposed to a solution of rare earth/heavy metalchelate labeled HSA. During the exposure of the section, the protein isexpected to interact specifically with the antibodies present on twofilaments (round areas on the section): the two filaments are thosebearing antibodies to HSA and the mixture of anti-HSA, Tf and Hp.Labeled HSA is not expected to interact with the filaments carryingantibodies to Tf alone or to the filament carrying streptavidin alone.

[0286] The sections are examined under an epifluorescence microscopeequipped with a 490 nm long pass filter or 600 nm bandpass filter forfluorescence visualization and a 35 mm camera or CCD camera. Excitationis carried out at 300 to 310 nm.

[0287] The sections then are washed extensively in PBS and reexaminedunder the fluorescence microscope. The resulting images are captured on35 mm color slides or CCD camera (digitized at about 1024×1024 pixelwith 12- or 16-grey bit scale levels assigned per pixel).

EXAMPLE 16 In Situ Solid Phase Synthesis of Polypeptides

[0288] Briefly, polystyrene beads (10-50 microns in diameter) from solidphase polypeptide synthesis with single amino acids covalently attachedare suspended in buffer and packed into hollow glass fibers of 500microns internal diameter under hydrostatic pressure initially and thenunder air pressure up to 500 psi to expel the supporting liquid. Thefiber then is heated briefly under controlled conditions to partiallysinter the contents. A solution for the amino acid subunits in asuitable solvent (e.g., N-methylpyrrolidine (NMP), dimethyl formamide(DMF), dichloromethane (methylene chloride) or chloroform) is mixed withthe particles. The subunits are preferably N-protected amino acids,typically one of the 20 naturally occurring L-amino acids havingprotected α-amine groups, and protected carboxy, hydroxy, thiol andamine side chain groups. The subunit molecules infiltrate the matricesof particles until substantially all of the subunits are entrapped in(or associated with) the particles. N-α-protected amino acids are addedto synthesize the peptide in 9-fluorenylmethoxycarbonyl (Fmoc) solvatedin DMF. Activated forms of the amino acids can be added as symmetricalanhydrides, pentafluorophenyl esters and1-oxo-2-hydroxydihydrobenzotriazine active esters. Alternatively,activating agents may be added to the polymer composition with thesolvent. A useful activating agent for Fmoc-based synthesis ishydroxy-O-benzotriazole, tetramethyluronium hexafluorophosphate (HBTU),to which is added hydroxy-O-benzotriazole (HOBT) anddiiospropylethylamine (DIEA). For Fmoc-based peptide synthesis,dimethylformamide (DMF)/N-methyl-pyrrolidone (NMP)/dimethylsulfoxide(DMSO) can be used.

[0289] The Fmoc protection group is first removed with piperidine andDNF, with the piperidine being thoroughly removed before addition of thenext residue. A solution of HBTU, HOBT, DMSO, NMP and DIEA is mixed withthe Fmoc amino acid to activate the amino acid to form a derivativewhich will react with the α-amino group of the growing peptide chainimmobilized on the fiber. The fiber is then washed with DMF and themixture containing the activated amino acid is flowed through the fiberto couple the amino acid subunit to the growing peptide chain. Aftercoupling, the Fmoc protection group is removed and the above procedureis repeated for subsequent amino acids until the planned peptide iscomplete.

[0290] An array of fibers then is prepared following the methods in theexamples above, embedded in a low viscosity epoxy resin withintermittent vacuum to remove air bubbles and then allowed to set. Thebundle is sectioned using a diamond saw. The array is used in aflowthrough arrangement so that the materials thereon can be manipulatedin a fashion similar to that conducted with larger multiwell microtiterplates as described in U.S. Pat. No. 5,843,767, supra.

EXAMPLE 17 Preparation of Omniclonal Antibodies Immobilized onControlled Pore Glass

[0291] Omniclonal antibody to human serum albumin and Omniclonalantibody to alpha-2-macroglobulin (BioSite Diagnostics, San Diego,Calif.) were immobilized on controlled pore glass (505 A, 200-400 mesh,CPG, Inc., Fairfield, N.J.) by adsorption from a solution containing apolyurethane polymer (Hypol® G-50 Prepolymer, The Dow Chemical Company,Midland, Mich.). CPG (200 mg) was placed in a glass screw cap vial and3.0 ml of a solution containing 20.5 mg Omniclonal antibody in 20 mMborate buffer, pH 8, and 0.4 ml of G-50 (10% in dry 2-propanol) wasadded. The samples were mixed gently for 3 hours at room temperature.The CPG particles were then washed by sedimentation 4 times with 5 ml ofborate buffer then dried at 4° C. The amount of protein bound to theparticles (30+/−5 mg/gm CPG ) was determined by absorbance at 280 nm.The coated particles were tested for antibody activity by incubationwith fluorescein-antigen for 1 hour, washed, and then viewed in afluorescent microscope. Only those coated particles treated with cognatefluorescent antigens bound fluorescent antigens.

EXAMPLE 18 Preparation of an Antibody Array

[0292] In this example, Omniclonal antibody to human serum albumin(Omni-HSA) and Omniclonal antibody to alpha-2-macroglobulin (Omni-α2M)were immobilized onto controlled pore glass. The particles weresuspended in a solution of 7.5% acrylamide with ammonium persulfate andaspirated into polymethyl methacrylate(PMMA) capillary tubing (1000μOD×750μ ID, Paradigm Optics, Inc., Pullman Wash.) and allowed to gel.Short lengths (1.5 cm) of each were then transferred to BEEM capsules(Polysciences, Inc. Warrington, Pa.) and a solution containing 80%methyl methacrylate (MMA), 20% butyl methacrylate (BMA) and 0.5% benzoinmethyl ether was added to fill the tubes. The tubes were capped andexposed to UV irradiation at 1 cm for 2.5 hours at room temperature,then allowed to cure overnight at room temperature. Thin sections (25μ)were prepared, mounted on glass slides and treated withfluorescein-human serum albumin (0.1 mg/ml in PBS containing 1 mg/mlBSA). The sections were then washed with PBS containing 0.5% Tween 20and viewed in a fluorescent microscope. Only samples containing CPG withOmni-HSA exhibited significant fluorescence.

EXAMPLE 19 Immobilization of Antibody in a Methacrylate Polymer withRetention of Antibody Activity

[0293] A cold mixture comprised of 1 part aqueous Omniclonal antibody(Omni-HSA and Omni-α₂M) and 10 parts methyl methacrylate(MMA)/polyethylene glycol methacrylate (PEGMA)/polyethylene glycoldimethacrylate (PEGDMA) (1:1:1) was vortexed vigorously then polymerizedby UV irradiation at 1 cm for 1 hour then cured overnight at 4° C. Theresulting blocks were not clear in appearance, but rather had many smallglobules suspended throughout the block. Thin sections (25μ) wereprepared, mounted on glass slides and treated with fluorescent antigen.The globules apparently resulted from a phase separation in which theaqueous, antibody-containing phase became suspended in the acrylatepolymer. Furthermore, the antibodies in the globules retained theiractivity, as demonstrated by their ability to specifically bindfluorescent antigen.

EXAMPLE 20 Immobilization of Antibody in a Methacrylate Polymer BySonication and Retention of Antibody Activity

[0294] In an attempt to disperse the globules in a more homogeneousfashion, a 10% suspension of Omniclonal antibodies in MMA/PEGMA/PEGDMA(1:1:1) was sonicated for two minutes. The suspension was then aspiratedinto PMMA capillary tubing (1000μ OD×750μ ID) and polymerized by UVirradiation. The tubing was then embedded in MMA/BMA by UV irradiationat 1 cm for 1 hour at room temperature. The resulting blocks weresectioned and the sections treated with fluorescein-HSA. For thisexperiment, both Omniclonal anti-HSA and Omniclonal α₂M were used.

EXAMPLE 21 Preparation of an Antibody Array

[0295] An antibody array was prepared as in Example 2 except the CPGparticles with immobilized Omniclonal antibodies were packed in PMMAcapillary tubings with MMANPEGMA/PEGDMA as binder, then the tubings werearranged in hexagonal patterns and embedded in MMA/BMA to form smallcylinders. Two unit devices were prepared in which experimental (filledcircles) and control (open circles) samples were arranged as shown inthe following schematic diagram.

[0296] Thin sections (25μ) of both of these units were prepared andtreated with fluorescein-HSA.

[0297] It will be understood that various modifications may be made tothe embodiments disclosed herein. Therefore, the above descriptionshould not be construed as limiting, but merely as exemplifications ofpreferred embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.

[0298] All patents and references cited herein are explicitlyincorporated by reference in their entirety.

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[0327] Patents

[0328] U.S. Pat. No. 5,843,767 Microfabricated, flow-through porousapparatus for discrete detection of binding reactions.

[0329] U.S. Pat. No. 4,289,623 Hollow fiber dialysis

[0330] U.S. Pat. No. 3,976,576 Dialyzer cartridge—Also, use of dialyzercartridge by filling hollow fibers and embed protein in fibers as theyare formed before the cartridges are cut.

What is claimed is:
 1. A fiber bundle comprising a plurality of fibersattached to each other in a fixed position with respect to each otherwherein the fibers have different agents of interest immobilized in oron different fibers.
 2. The fiber bundle according to claim 1 comprisingat least 100 different fibers.
 3. The fiber bundle according to claim 1wherein the agent of interest is selected from the group consisting of amicroorganism, ligand, antibody, antigen, nucleic acid, polysaccharide,receptor, plant or animal cells, organelles and fractions thereof. 4.The fiber bundle according to claim 1 further comprising a plurality ofsolid phases immobilizing said agents of interest wherein said solidphase is immobilized in or on the fibers.
 5. The fiber bundle accordingto claim 4 wherein said solid phases are particles or thread-likestructures embedded in the fibers.
 6. The fiber bundle according toclaim 1 wherein all or most of the fibers contain a differentimmobilized agent of interest.
 7. The fiber bundle according to claim 1wherein at least one of the fibers contains a dye.
 8. The fiber bundleaccording to claim 1 wherein different fibers contain differentconcentrations of the agent of interest.
 9. The fiber bundle accordingto claim 1 wherein each fiber contains no more than one immobilizedagent of interest.
 10. A method of forming the bundle of claim 1comprising a) immobilizing different agents of interest in or ondifferent fibers, b) aligning the fibers in a fiber bundle, and c)fixing the arrangement of fibers in the fiber bundle.
 11. The method ofclaim 10 wherein said immobilizing comprises mixing an agent of interestin a liquid and solidifying the liquid to form a fiber.
 12. The methodof claim 11 wherein a liquid mixture of the agent of interest is castinto a fiber.
 13. The method of claim 11 wherein the liquid contains apolymer gelling material.
 14. The method of claim 11 wherein the liquidcontains a polymerizable monomer.
 15. The method of claim 10 whereinsaid immobilizing comprises immobilizing an agent of interest to apreformed fiber.
 16. A method for making an array comprising forming thefiber bundle of claim 1 and cutting the fiber bundle transversely or atan angle to form a section such that the fixed position with respect toeach other is maintained.
 17. The method of claim 16 further comprisingmounting said section to a solid support to form an array.
 18. Themethod of claim 16 wherein said sections are less than 1 cm thick. 19.An array comprising a plurality of cells in a known location on thearray, each cell containing an agent of interest immobilized in or on atleast a portion of a fiber, wherein different cells contain differentfibers or portions of a fiber which contains a different agent ofinterest immobilized therein or thereon, and wherein each agent ofinterest is located in a known cell.
 20. The array of claim 19 whereinthe array contains portions of each fiber prepared by cutting a sectionfrom said fiber.
 21. The array of claim 20 wherein the cells eachcontain one well or channel.
 22. An array prepared by the method ofclaim
 16. 23. An array prepared by the method of claim
 17. 24. An arrayprepared by the method of claim
 18. 25. A binding assay for detecting ananalyte in a sample wherein said anaiyte binds to at least one agent ofinterest in an array comprising; contacting a samnple suspected ofcontaining an analyte with the array of claim 19 under conditionspermitting the binding of analyte to agent of interest, detecting thepresence or absence of binding between analyte and each cell in thearray, and determining the presence or absence of the analyte by thepresence of any binding being detected at a predetermined cell of thearray.
 26. The binding assay of claim 25, further comprising; adding alabeled detection agent capable of binding to cells having eitheranalyte bound to agent of interest or cells not having the analyte sobound, but not both, and detecting the presence of the labeled detectionagent in one or more cells of the array.
 27. A binding assay fordetecting an analyte in a sample wherein said analyte binds to at leastone agent of interest in an array comprising; contacting a samplesuspected of containing an analyte with the array of claim 22 underconditions permitting the binding of analyte to agent of interest,detecting the presence or absence of binding between analyte and eachcell in the array, and determining the presence or absence of theanalyte by the presence of any binding being detected at a predeterminedcell of the array.
 28. The binding assay of claim 27, further comprisingadding a labeled detection agent capable of binding to cells havingeither analyte bound to agent of interest or cells not having theanalyte so bound, but not both, and detecting the presence of thelabeled detection agent in one or more cells of the array.
 29. A methodof determining whether the fibers in the bundle of claim 1 are alignedcomprising illuminating fibers individually at one end of the bundle andphotoelectrically identifying the location of a signal at the other endof the bundle.
 30. A microarray comprising a solid phase support and atleast about 500 cells per square centimeter wherein each cell contains aagent of interest that is not chemically bound to the solid phasesupport.
 31. The microarray of claim 30 containing at least about 1,000cells per square centimeter.
 32. A microarray comprising a solid phasesupport and at least about 500 cells per square centimeter wherein eachcell contains an agent of interest which is a macromolecule, amicroorganism, a plant or animal cell, an organelle or a fraction of abiological cell.
 33. The microarray of claim 32 containing at leastabout 1,000 cells per square centimeter.
 34. A microarray comprising asolid phase support and at least about 500 cells per square centimeterwherein each cell contains an agent of interest that was synthesizedprior to contacting the solid phase support.
 35. The microarray of claim34 containing at least about 1,000 cells per square centimeter.
 36. Amicroarray comprising a solid phase support and at least about 500different cells per square centimeter wherein each cell is formed by asolid material adhered to said solid phase support wherein each solidmaterial contains a agent of interest.
 37. The microarray of claim 36containing at least about 1,000 cells per square centimeter.
 38. Amultiwell plate containing at least about 500 wells per squarecentimeter.
 39. The multiwell plate of claim 38 wherein walls of thewells are made of a heterologous material from a base of the well.
 40. Athin elongated fiber impregnated with a solid phase wherein the solidphase is bound to a agent of interest.
 41. A solid phase constructcontaining an immobilized agent of interest comprising an embeddingmedium, a porous or hollow solid phase and a agent of interest, whereinthe agent of interest is immobilized on inside surfaces of the porous orhollow solid phase, wherein the porous or hollow solid phase is embeddedin the embedding medium, and wherein the inside surfaces are exposed tothe surface of the construct by cleaving such that individual porous orhollow solid phases are cleaved in plural sections.
 42. A microarraycontaining a plurality of different cells wherein each cell contains asolid phase support, a porous particle containing a agent of interestimmobilized on an inside surface of a porous particle and a medium forattaching said particle to said solid phase support in a particularcell.
 43. The microarray of claim 42 wherein the porous particle hasbeen cleaved to expose agents of interest on inner surfaces of theporous particle.
 44. An elongated fiber having an agent of interestimmobilized thereon or therein such that a detectable number of singleagents of interest are present in each millimeter of fiber length.
 45. Across-section of the fiber of claim 44 containing a detectable number ofthe agent of interest.
 46. A fibrous structure comprising; at least twofibers of claim 44 being fixed in parallel juxtaposition to each other,and at least two agents of interest being immobilized in or on thefibers, wherein each fiber contains a different agent of interest. 47.The fibrous structure of claim 46 wherein each of said at least twofibers contains one but not the other agent of interest.
 48. The fibrousstructure of claim 46 wherein at least 10 different fibers are present.49. The fibrous structure of claim 48 wherein each fiber contains onlyone agent of interest, being substantially free of other agents ofinterest.
 50. The fibrous structure of claim 46 wherein each fibercontains a mixture of plural agents of interest and each fiber containsa different mixture of plural different agents of interest.
 51. A fibercross-section structure comprising a cross-section of at least twofibers, each fiber being set in parallel juxtaposition to another fiber,and at least two agents of interest being immobilized in or on thefibers, wherein each fiber contains a different agent of interest. 52.The fiber cross-section structure of claim 51 wherein each of said atleast two fibers contains one but not the other agent of interest. 53.The fiber cross-section structure of claim 51 wherein at least 10different fibers are present.
 54. The fiber cross-section structure ofclaim 53 wherein each fiber contains only one agent of interest, beingsubstantially free of other agents of interest.
 55. The fibercross-section structure of claim 51 wherein each fiber contains amixture of plural agents of interest and each fiber contains a differentmixture of plural different agents of interest.
 56. A microarraycomprising a solid block on an inert solid support, the solid blockcontaining a plurality of cells exposed to the surface of the blockwherein each cell contains a different agent of interest and wasindependently prepared and embedded into the solid block before mountingon the inert solid support.
 57. The microarray of claim 56 wherein theblock is less than 50 μm thick.
 58. The microarray of claim 57 whereinthe block is less than 20 μm thick.
 59. The microarray of claim 56wherein the block contains at least 100 cells.
 60. The method of claim10 further comprising assaying each of said fibers for the presence ofeach of the different agents of interest before said aligning the fibersin a fiber bundle.
 61. A method of forming the bundle of claim 1,comprising: aligning the fibers in a fiber bundle; passing liquidscontaining different agents of interest through or contacting differentfibers and fixing or allowing the different agents of interest to becomefixed in or on fibers in the fiber bundle.
 62. The method of claim 61,wherein said agent of interest in said liquid is immobilized bysolidifying the liquid.
 63. The method of claim 62, wherein the liquidcontains a polymer gelling material.
 64. The method of claim 62, whereinthe liquid contains a polymerizable monomer.
 65. The method of claim 61,wherein said agent of interest is immobilizing on or in a preformedfiber.
 66. A microarray comprising: a solid phase having a surface, anda plurality of structures bound to said surface, wherein each of aplurality of structures has an immobilized agent of interest availablefor binding to a target, wherein the target is a binding partner for theagent of interest, and wherein the microarray contains a plurality ofdifferent agents of interest in a corresponding plurality of differentstructures.
 67. The microarray of claim 66, wherein the structures arepermeable to the target.
 68. The microarray of claim 67, wherein thestructures are a gel.
 69. The microarray of claim 66, wherein thestructures are made of impermeable material and the agent of interest isimmobilized on a surface thereof.
 70. The microarray of claim 69,wherein each of the structure forms a hollow chamber with the agent ofinterest immobilized on an inner surface of the hollow chamber.
 71. Amicroarray comprising: a solid phase having a flat surface, and aplurality of structures projecting away from the plane of the solidphase, wherein each of a plurality of the structures an agent ofinterest, wherein the agent of interest is available for binding to atarget in a sample applied to the microarray, wherein the target is abinding partner for the agent of interest, and wherein the microarraycontains a plurality of different agents of interest in a correspondingplurality of different structures.
 72. The microarray of claim 71,wherein the structures are made of impermeable material and the agent ofinterest is immobilized on a surface thereof.
 73. The microarray ofclaim 71, wherein solid material between the structures is removed. 74.A method of manufacturing a microarray device comprising: synthesizingat least one functional moiety onto a plurality of fibers wherein one ormore fibers receives at least one moiety, bundling said plurality offibers in a predetermined arrangement; bonding or fixing said bundledplurality of fibers to fix the positions of the fibers; and cleavingsaid bundled fibers into a plurality of chips to be deposited at aspecific address on a solid phase.
 75. The method of claim 74, whereinsaid at a least one functional moiety is selected from the groupconsisting of DNA, oligonucleotides, proteins, peptides,polysaccharides, lipids, carbohydrates and small organic compounds. 76.The method of claim 74, wherein said fibers are comprised of aheterogeneous matrix of at least two or more materials having dissimilarphysicochemical properties.
 77. The method of claim 76, wherein saidheterogeneous matrix comprises a mixture of materials selected from thegroup consisting of plastics, hydrogels, glass fibers, wax, clay,colloid suspensions, alginates, and dextrans.
 78. The method of claim76, wherein at least one of the materials comprising said heterogeneousmatrix receives at least one functional moiety.
 79. The method of claim78, wherein said at a least one functional moiety is selected from thegroup consisting of DNA, oligonucleotides, proteins, peptides,polysaccharides, lipids, carbohydrates and small organic compounds. 80.The method of claim 79, wherein said at least one functional moiety is aprotein.